Non-invasive prenatal diagnosis of fetal genetic condition using cellular DNA and cell free DNA

ABSTRACT

Disclosed are methods for determining at least one sequence of interest of a fetus of a pregnant mother. In various embodiments, the method can determine one or more sequences of interest in a test sample that comprises a mixture of fetal cellular DNA and mother-and-fetus cfDNA. In some embodiments, methods are provided for determining whether the fetus has a genetic disease. In some embodiments, methods are provided for determining whether the fetus is homozygous in a disease causing allele when the mother is heterozygous of the same allele. In some embodiments, methods are provided for determining whether the fetus has a copy number variation (CNV) or a non-CNV genetic sequence anomaly.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims benefits under 35 U.S.C. § 119(e) to U.S.Provisional Patent Application No. 62/026,548, entitled: NON-INVASIVEPRENATAL DIAGNOSIS OF FETAL GENETIC CONDITION USING CELLULAR DNA ANDCELL FREE DNA, filed Jul. 18, 2014, which is herein incorporated byreference in its entirety for all purposes.

BACKGROUND

The determination of copy number of genetic sequences in a fetus is ofimportant diagnostic value. For instance, in a dominant geneticdisorder, the presence of a single copy of a disease causing allelecauses the phenotypical expression of the genetic disorder. In contrast,in a recessive genetic disorder, the presence of a single copy of adisease causing allele only renders the individual a carrier, and doesnot cause the phenotypical expression of the genetic disorder. Inaddition, abnormal copy numbers of genetic sequences, e.g., chromosomesegments or whole chromosomes in partial or complete aneuploidy, oftencause various genetic disorders. For instance, trisomy 21 causes DownSyndrome (DS).

Previously, most information about copy number and copy number variation(CNV) of the fetus was provided by cytogenetic resolution that haspermitted recognition of structural abnormalities. Conventionalprocedures for genetic screening and biological dosimetry have utilizedinvasive procedures, e.g., amniocentesis, cordocentesis, or chorionicvillus sampling (CVS), to obtain fetal cells for the analysis ofkaryotypes. Recognizing the need for more rapid testing methods that donot require cell culture, fluorescence in situ hybridization (FISH),quantitative fluorescence PCR (QF-PCR) and array-Comparative GenomicHybridization (array-CGH) have been developed as molecular-cytogeneticmethods for the analysis of copy number variations. The advent oftechnologies that allow for sequencing entire genomes in relativelyshort time, and the discovery of circulating cell-free DNA (cfDNA)including both maternal and fetal DNA in the pregnant mother's bloodhave provided the opportunity to analyze fetal genetic materials withoutthe risks associated with invasive sampling methods, which provides atool to diagnose various kinds of copy number variation (CNV) of geneticsequences of interest.

Diagnosis of copy number variation (CNV) in some applications involvesheightened technical challenges. When the mother is a carrier for arecessive genetic disease, the fetus has a 25% chance of developing thegenetic disease if the father is also a carrier. In such case, themother is heterozygous of the disease related gene, having one diseasecausing allele and one normal allele; the fetus is homozygous of thedisease related gene, having two copies of the disease causing allele.It is desirable to determine if the fetus has inherited geneticdisease-causing mutated alleles from both parents in a non-invasivemanner using maternal plasma cfDNA. However, it is difficult todifferentiate if the fetus is homozygous or heterozygous when the motheris heterozygous using conventional method of non-invasive prenataldiagnosis (NIPD) because the two scenarios have similar sequence tagsmapping to the two alleles for a biallelic gene. Furthermore, some NIPDmethods only use polymorphism sequences from homozygous mother andheterozygous fetus to determine fetal zygosity and fetal fraction. Suchapproach cannot use genetic materials from heterozygous mother todetermine fetal zygosity and fetal fraction, therefore limiting thesensitivity and/or efficiency of diagnosis. These challenges underliethe continuing need for noninvasive methods that would reliably diagnosecopy number in a variety of clinical settings. Embodiments disclosedherein fulfill some of the above needs and in particular offer anadvantage in providing a reliable method that is applicable to thepractice of noninvasive prenatal diagnostics.

SUMMARY

In some embodiments, methods are provided for determining the presence,abundance, or copy number of a sequence of interest in the fetus, e.g.,a clinically relevant sequence, using fetus-only cellular DNA and cfDNAincluding maternal and fetal DNA. In some embodiments, methods areprovided for determining whether the fetus has a genetic disease, ormore specifically, for determining whether the fetus is homozygous in adisease causing allele when the mother is heterozygous of the sameallele.

In specific embodiments, the invention provides methods of reliablyestimating fetal fraction from polymorphisms such as small basevariations or insertions-deletions which are robust with respect toparental ethnicity, embryo sex, gestational age and other environmentalfactors.

In some embodiments, methods are provided for determining copy numbervariation (CNV) of partial or complete fetal aneuploidy. CNV that can bedetermined according to the present method include trisomies andmonosomies of any one or more of chromosomes 1-22, X and Y, otherchromosomal polysomies, and deletions and/or duplications of segments ofany one or more of the chromosomes.

One aspect of the disclosure provides a method for determining at leastone sequence of interest of a fetus of a pregnant mother, the methodinvolves: (a) obtaining cellular DNA from the blood of the pregnantmother, where the cellular DNA includes fetal cellular DNA; (b)obtaining mother-and-fetus mixed cfDNA from the blood of the pregnantmother; (c) applying an indicator to at least one of the fetal cellularDNA and the mixed cfDNA, wherein the indicator identifies a source ofDNA as being from the fetal cellular DNA or the mixed cfDNA; (d)combining the fetal cellular DNA and the mixed cfDNA to provide a sampleof combined cellular DNA and cfDNA; (e) sequencing the sample ofcombined cellular DNA and cfDNA to provide a plurality of sequence tags;and (f) analyzing the plurality of sequence tags to determine thepresence and/or abundance of the at least one sequence of interest inthe fetus's DNA, where at least a portion of the plurality of sequencetags map to the at least one sequence of interest.

Implementations may include one or more of the following features. Insome implementations, (e) sequencing said sample of combined cellularand cfDNA involves: sequencing said sample of combined cellular andcfDNA to produce a plurality of sequence reads; and aligning theplurality of sequence reads to a reference sequence to provide theplurality of sequence tags, where sources of the plurality of sequencetags are indicated by the indicator identifying the source of DNA.

In some implementations, the fetal cellular DNA is obtained from one ormore fetal nucleated red blood cells (NRBCs) in the blood of thepregnant mother. The method further involves separating the fetal NRBCsfrom maternal erythrocytes in a cellular component of a blood sample ofthe pregnant mother. In some implementations, separating the fetal NRBCsfrom the maternal erythrocytes involves differentially lysing maternalerythrocytes.

In some implementations, separating the fetal NRBCs from the maternalerythrocytes includes size-based separation and/or capture-basedseparation. In some implementations, the capture-based separationinvolves capturing the fetal NRBCs through binding one or more cellularmarkers expressed by fetal NRBCs. In some implementations, the one ormore cellular markers expressed by fetal NRBCs are selected from thegroup including CD71, CD36, CD34, antigen-i, galactose, glycophorin-a,fetal haemoglobin, and any combinations thereof. In someimplementations, the one or more cellular markers include a surfacemarker expressed by fetal NRBCs but not, or to a lesser degree, bymaternal NRBCs. In some implementations, the one or more cellularmarkers includes a 4B9-antigen and/or a 4B8-antigen. In someimplementations, the capture-based separation involves bindingmagnetically responsive particles to fetal NRBCs, where the magneticallyresponsive particles have an affinity to one or more cellular markersexpressed by fetal NRBCs. In some implementations, the capture-basedseparation is performed by an automated immunomagnetic separationdevice. In some implementations, the capture-based separation involvesbinding fluorescent labels to fetal NRBCs, where the fluorescent labelshave an affinity to one or more cellular markers expressed by fetalNRBCs.

In some implementation, the method further involves: obtaining a bloodsample from the pregnant mother. The method may also involve separatingan erythrocyte fraction and a plasma fraction of the blood sample. Themethod may also involve obtaining the fetal cellular DNA from theerythrocyte fraction of the blood sample. The method may also involveobtaining the cfDNA from the plasma fraction of the blood sample. Themethod further involves preparing a first sequencing library of thefetus-only cellular DNA and a second sequencing library of the cfDNA,where applying the indicator in (c) comprises incorporating indexes ineach of said sequencing libraries, wherein the indexes incorporated insaid first library differ from the indexes incorporated in said secondlibrary, and the indexes are identifiable from said plurality ofsequence tags.

In some implementations, incorporating indexes in each of saidsequencing libraries involves hybridizing and extending adapteroligonucleotides including the indexes. In some implementations, theadapter oligonucleotides include locus-specific extensionoligonucleotides. In some implementations, the locus-specific extensionoligonucleotides are selective for two or more alleles of a diseaserelated gene. In some implementations, each of the adapteroligonucleotides includes an adapter sequence or a portion thereof,where the adapter sequence is configured to hybridize to anoligonucleotide attached to a substrate of a flow cell of a sequencingapparatus. In some implementations, incorporating indexes to each ofsaid sequencing libraries involves ligating or transposing sequencesincluding the indexes to the fetal cellular DNA and the mixed cfDNA. Themethod further involves incorporating an individual-specific index tothe sequencing libraries, where the individual-specific index indicatesthe identity of the pregnant mother, thereby allowing the pregnantmother's DNA to be processed with other individuals' DNA for parallelsequencing. In some implementations, the sequencing libraries aretransposon insertion libraries.

In some implementations, the method further involves determining whetherthe fetus has a genetic disease from the at least one sequence ofinterest of the fetus. In some implementations, the at least onesequence of interest includes a disease associated allele selected fromthe group including: a single nucleotide polymorphism, a tandem repeat,a micro-deletion, an insertion, an indel, and any combinations thereof.

In some implementations, the method further involves enriching thesequence of interest using a primer including a locus-specific extensionoligonucleotide that hybridizes to two or more alleles of a gene relatedto the disease. In some implementations, the primer further includes anindex sequence. In some implementations, the method further involvesenriching the sequence of interest applying two primer sequencesbracketing the sequence of interest. In some implementations, the atleast one sequence of interest includes a chromosome or a chromosomesegment. In some implementations, the method further involvesdetermining a complete or partial aneuploidy.

Another general aspect of the disclosure provides a method, implementedat a computer system that includes one or more processors and systemmemory, for determining a condition of a fetus related to a sequence ofinterest. The method involves obtaining, by the computer system,sequence reads of fetus-only cellular DNA obtained from a blood sampleof the mother carrying the fetus, the cellular DNA having beenspecifically enriched for a sequence of interest; computing, by thecomputer system, a count of sequence tags mapping to the sequence ofinterest for the cellular DNA; obtaining, by the computer system,sequence reads of mother-and-fetus mixed cfDNA obtained from the mother,the cfDNA having been specifically enriched for the sequence ofinterest; computing, by the computer system, a count of sequence tagsmapping to the sequence of interest for the cfDNA; comparing, by thecomputer system, the sequence tag counts mapping to the sequence ofinterest between the cellular DNA and the cfDNA; and determining, by thecomputer system, the condition of the fetus related to the sequence ofinterest. In some implementations, the specifically enriched cellularDNA and the specifically enriched cfDNA were combined for amplificationand/or sequencing.

Computer program products and systems implementing the methods describedabove are also provided.

Although the examples herein concern humans and the language isprimarily directed to human concerns, the concepts described herein areapplicable to genomes from any plant or animal. These and other objectsand features of the present disclosure will become more fully apparentfrom the following description and appended claims, or may be learned bythe practice of the disclosure as set forth hereinafter.

INCORPORATION BY REFERENCE

All patents, patent applications, and other publications, including allsequences disclosed within these references, referred to herein areexpressly incorporated herein by reference, to the same extent as ifeach individual publication, patent or patent application wasspecifically and individually indicated to be incorporated by reference.All documents cited are, in relevant part, incorporated herein byreference in their entireties for the purposes indicated by the contextof their citation herein. However, the citation of any document is notto be construed as an admission that it is prior art with respect to thepresent disclosure.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a process of a method for determining a condition of afetus related to a sequence of interest involving analyses ofmother-only cellular DNA.

FIG. 2 shows a process of a method for determining conditions related toa sequence of interest for a fetus using a blood sample from thepregnant mother including mother-only cellular DNA.

FIG. 3 shows a diagram of a workflow according to some embodimentsinvolving analyses of mother-only cellular DNA, wherein theincorporation of index sequences uses two primers to introduce twoadaptor segments.

FIG. 4 shows a workflow that uses only one primer when incorporatingindexes, and introduces only one sequencing adaptor (P5) instead of twoadaptors.

FIGS. 5A and 5B describe a workflow where the mother-only cellular DNAand mixture cfDNA are indexed independently, mixed together, and thensubjected to the multiplexed SNP-interrogation at the same time in asingle tube.

FIG. 6 is a flowchart showing a process for determining a sequence ofinterest for the fetus using fetal cellular DNA and mother-and-fetuscfDNA according to some embodiments of the disclosure.

FIG. 7 is a flowchart showing a process of obtaining mother-and-fetuscfDNA and fetal cellular DNA using fixed blood.

FIG. 8 is a flowchart showing a process of obtaining mother-and-fetuscfDNA and fetal cellular DNA using unfixed blood.

FIG. 9 is a flowchart showing a process for isolating fetal nucleatedred blood cells (fetal NRBCs) from a maternal blood sample according tosome embodiments.

FIG. 10 is a flowchart showing a process for preparing an indexedlibrary using fetal cellular DNA.

FIG. 11 is a flowchart showing a process for preparing an indexedlibrary using mother-and-fetus cfDNA.

FIG. 12 shows a diagram of a workflow according to some embodiments ofthe invention, wherein the incorporation of index sequences occur withtwo primers introducing two adaptor segments.

FIG. 13 shows a process for determine fetal zygosity of a sequence ofinterest using mother-only cellular DNA and mother-and-fetus cfDNA.

FIGS. 14A-14D illustrates a hypothetical example of some embodimentsinvolving analyses of mother-only cellular DNA and mother-and-fetuscfDNA and a comparison to a conventional method.

FIG. 15 is a block diagram of a dispersed system for processing a testsample and ultimately making a diagnosis.

FIG. 16 schematically illustrates how different operations in processingtest samples may be grouped to be handled by different elements of asystem.

FIGS. 17A and 17B show electropherograms of a cfDNA sequencing libraryprepared according to the abbreviated protocol described in Example 1a(FIG. 17A), and the protocol described in Example 1b (FIG. 17B).

FIG. 18 shows the relative frequency of sequence length for fourlibraries made from purified cfDNA according to one example of thedisclosure.

FIG. 19 shows the test results of the Y chromosome specific gene sry for9 samples using fetal cellular DNA and cfDNA.

DETAILED DESCRIPTION Definitions

Unless otherwise indicated, the practice of the method and systemdisclosed herein involves conventional techniques and apparatus commonlyused in molecular biology, microbiology, protein purification, proteinengineering, protein and DNA sequencing, and recombinant DNA fields,which are within the skill of the art. Such techniques and apparatus areknown to those of skill in the art and are described in numerous textsand reference works (See e.g., Sambrook et al., “Molecular Cloning: ALaboratory Manual,” Third Edition (Cold Spring Harbor), [2001]); andAusubel et al., “Current Protocols in Molecular Biology” [1987]).

Numeric ranges are inclusive of the numbers defining the range. It isintended that every maximum numerical limitation given throughout thisspecification includes every lower numerical limitation, as if suchlower numerical limitations were expressly written herein. Every minimumnumerical limitation given throughout this specification will includeevery higher numerical limitation, as if such higher numericallimitations were expressly written herein. Every numerical range giventhroughout this specification will include every narrower numericalrange that falls within such broader numerical range, as if suchnarrower numerical ranges were all expressly written herein.

The headings provided herein are not intended to limit the disclosure.

Unless defined otherwise herein, all technical and scientific terms usedherein have the same meaning as commonly understood by one of ordinaryskill in the art. Various scientific dictionaries that include the termsincluded herein are well known and available to those in the art.Although any methods and materials similar or equivalent to thosedescribed herein find use in the practice or testing of the embodimentsdisclosed herein, some methods and materials are described.

The terms defined immediately below are more fully described byreference to the Specification as a whole. It is to be understood thatthis disclosure is not limited to the particular methodology, protocols,and reagents described, as these may vary, depending upon the contextthey are used by those of skill in the art.

As used herein, the singular terms “a,” “an,” and “the” include theplural reference unless the context clearly indicates otherwise.

Unless otherwise indicated, nucleic acids are written left to right in5′ to 3′ orientation and amino acid sequences are written left to rightin amino to carboxy orientation, respectively.

“Whole blood sample” herein refers to a whole blood sample that has notbeen fractionated or separated into its component parts. Whole blood isoften combined with an anticoagulant such as EDTA or ACD during thecollection process, but is generally otherwise unprocessed. In the US,the capitalized “Whole Blood” means a specific standardized product fortransfusion or further processing, where “whole blood” is any unmodifiedcollected blood.

“Blood fractionation” is the process of fractionating whole blood orseparating it into its component parts. This is typically done bycentrifuging the blood. The resulting components are: (a) a clearsolution of blood plasma in the upper phase (which can be separated intoits own fractions), (b) a buffy coat, which is a thin layer ofleukocytes (white blood cells) mixed with platelets in the middle, and(c) erythrocytes (red blood cells) at the bottom of the centrifuge tubein the hematocrit faction.

Serum separation tubes (SSTs) are tubes used in phlebotomy containing asilicone gel; when centrifuged the silicone gel forms a layer on top ofthe buffy coat, allowing the blood plasma to be removed more effectivelyfor testing and related purposes.

“Blood plasma” or “plasma” is the straw-colored/pale-yellow liquidcomponent of blood that normally holds the blood cells in whole blood insuspension. It makes up about 55% of total blood by volume. It is theintravascular fluid part of [extracellular fluid] (all body fluidoutside of cells). It is mostly water (93% by volume), and containsdissolved proteins including albumins, immunoglobulins, and fibrinogen,glucose, clotting factors, electrolytes (Na+, Ca2+, Mg2+, HCO3− Cl−etc.), hormones and carbon dioxide.

Blood plasma can be prepared by centrifuging a tube of whole blood andcontaining an anticoagulant until the blood cells fall to the bottom ofthe tube. The blood plasma is then poured or drawn off. Blood plasma hasa density of approximately 1025 kg/m3, or 1.025 kg/l.

“Peripheral blood” is blood that obtained from acral areas, or from thecirculation remote from the heart; the blood in the systemiccirculation.

“Fixing” is a technique that maintains the structure of cells and/orsub-cellular components such as cell organelles (e.g., nucleus). Fixingmodifies the chemical or biological structure cellular components by,e.g., cross-linking them. Fixing may cause whole cells and cellularorganelles to resist lysis. Of interest, fixing may also cause cellularnucleic acids to resist release into a surrounding medium. For example,fixing may prevent nuclear DNA from white blood cells to resist releaseinto a plasma fraction during centrifugation of whole blood.

“Fixative” is an agent such as a chemical or biological reagent thatfixes cellular nucleic acids and thereby causes cells to resist releaseof such nucleic acids into a surrounding medium. A fixative may disablecellular proteolytic enzymes and nucleases. Examples of fixativesinclude aldehydes (e.g., formaldehyde), alcohols, and oxidizing agents.Examples of suitable fixatives are presented in US Patent ApplicationPublication 2010/0184069, filed Jan. 19, 2010, and in US PatentApplication Publication No. 2010/209930, filed Feb. 11, 2010, eachincorporated herein by reference in its entirety. A vendor ofcommercially available fixative compositions for fixing nuclei of whiteblood cells is Streck, Inc. of Omaha Neb. Streck blood collection tubessuch the Streck Cell-free DNA BCT contain a mild preservative, whichfixes cellular nuclei and large cellular components, thereby inhibitingwhite blood cell lysis that can contaminate plasma DNA with cellularDNA.

The term “copy number variation” herein refers to variation in thenumber of copies of a nucleic acid sequence present in a test sample incomparison with the copy number of the nucleic acid sequence present ina reference sample. In certain embodiments, the nucleic acid sequence is1 kb or larger. In some cases, the nucleic acid sequence is a wholechromosome or significant portion thereof. A “copy number variant”refers to the sequence of nucleic acid in which copy-number differencesare found by comparison of a sequence of interest in test sample with anexpected level of the sequence of interest. For example, the level ofthe sequence of interest in the test sample is compared to that presentin a qualified sample. Copy number variants/variations includedeletions, including microdeletions, insertions, includingmicroinsertions, duplications, multiplications, and translocations. CNVsencompass chromosomal aneuploidies and partial aneuploidies.

The term “aneuploidy” herein refers to an imbalance of genetic materialcaused by a loss or gain of a whole chromosome, or part of a chromosome.

The terms “chromosomal aneuploidy” and “complete chromosomal aneuploidy”herein refer to an imbalance of genetic material caused by a loss orgain of a whole chromosome, and includes germline aneuploidy and mosaicaneuploidy.

The terms “partial aneuploidy” and “partial chromosomal aneuploidy”herein refer to an imbalance of genetic material caused by a loss orgain of part of a chromosome, e.g., partial monosomy and partialtrisomy, and encompasses imbalances resulting from translocations,deletions and insertions.

The term “plurality” refers to more than one element. For example, theterm is used herein in reference to a number of nucleic acid moleculesor sequence tags that is sufficient to identify significant differencesin copy number variations in test samples and qualified samples usingthe methods disclosed herein. In some embodiments, at least about 3×106sequence tags of between about 20 and 40 bp are obtained for each testsample. In some embodiments, each test sample provides data for at leastabout 5×106, 8×106, 10×106, 15×106, 20×106, 30×106, 40×106, or 50×106sequence tags, each sequence tag comprising between about 20 and 40 bp.

The terms “polynucleotide,” “nucleic acid” and “nucleic acid molecules”are used interchangeably and refer to a covalently linked sequence ofnucleotides (i.e., ribonucleotides for RNA and deoxyribonucleotides forDNA) in which the 3′ position of the pentose of one nucleotide is joinedby a phosphodiester group to the 5′ position of the pentose of the next.The nucleotides include sequences of any form of nucleic acid,including, but not limited to RNA and DNA molecules such as cfDNAmolecules. The term “polynucleotide” includes, without limitation,single- and double-stranded polynucleotide.

The terms “cell-free DNA” (cfDNA) and “circulating cell-free DNA” areused herein interchangeably to refer to fragments of DNA existingoutside of cells in vivo, for example, circulating in maternal blood.The terms can also be used to refer to the fragments of DNA that havebeen obtained from the in vivo extracellular sources and separated,isolated or otherwise manipulated in vitro. The fragments of cfDNAtypically have length ranging about 150-200 bp and averaging about 170bp, which presumably relates to the length of a DNA stretch wrappedaround a nucleosome. The cfDNA circulating in a pregnant woman includeDNA fragments from the mother and the fetus or fetuses, with the fetalcomponent ranging up to about 20% in some cases and scenarios, which isreferred to as fetal fraction. In many situations, fetal fraction isoften less than 20%. Similarly, the terms “cell-free RNA” and“circulating cell-free RNA” are used herein interchangeably to refer tofragments of RNA existing outside of cells in vivo, for example,circulating in maternal blood. Several embodiments of the methods andcompositions exemplified herein with regard to cfDNA can be used forcell-free RNA as well.

The terms “cellular DNA,” and “cellular genomic DNA,” are usedinterchangeably herein with reference to DNA existing in a cell in vivoand containing a complete genome of the cell or organism. The terms canalso be used to refer to DNA that has been obtained from the in vivocell and separated, isolated or otherwise manipulated in vitro so longas the DNA was not removed from the cell in vivo. Typically, the cell isremoved from cfDNA prior to the cell being lysed to produce in vitrocellular DNA. Although cfDNA theoretically may collectively constitute acomplete genome, the term gDNA as used herein does not include cfDNA.Similarly, the term “cellular RNA” is used herein to refer to RNAexisting in a cell in vivo. Several embodiments of the methods andcompositions exemplified herein with regard to cellular DNA can be usedfor cellular RNA as well.

The term “portion” is used herein in reference to the amount of sequenceinformation of fetal and maternal nucleic acid molecules in a biologicalsample that in sum amount to less than the sequence information of 1human genome.

The terms “index,” “index sequence,” “unique identifier,” “barcode,” and“barcode sequence” are used interchangeably herein unless specifiedotherwise. The terms refer to a sequence of nucleotides, usuallyoligonucleotides, that can be used to identify a sequence of interest.The index sequence may be exogenously incorporated into the sequence ofinterest by ligation, extension, or other methods known in the art. Theindex sequence may also be endogenous to the sequence of interest, e.g.,a fragment in the sequence of interest itself may be used as an index.For implementations of index sequences, see, Kinde, et al. (2011),Proceedings of the National Academy of Sciences, 108, 9530.

The term “test sample” herein refers to a sample, typically derived froma biological fluid, cell, tissue, organ, or organism, comprising anucleic acid or a mixture of nucleic acids comprising at least onenucleic acid sequence that is to be screened for copy number variation.In certain embodiments the sample comprises at least one nucleic acidsequence whose copy number is suspected of having undergone variation.Such samples include, but are not limited to sputum/oral fluid, amnioticfluid, blood, a blood fraction, or fine needle biopsy samples (e.g.,surgical biopsy, fine needle biopsy, etc.), urine, peritoneal fluid,pleural fluid, and the like. Although the sample is often taken from ahuman subject (e.g., patient), the assays can be used to copy numbervariations (CNVs) in samples from any mammal, including, but not limitedto dogs, cats, horses, goats, sheep, cattle, pigs, etc. The sample maybe used directly as obtained from the biological source or following apretreatment to modify the character of the sample. For example, suchpretreatment may include preparing plasma from blood, diluting viscousfluids and so forth. Methods of pretreatment may also involve, but arenot limited to, filtration, precipitation, dilution, distillation,mixing, centrifugation, freezing, lyophilization, concentration,amplification, nucleic acid fragmentation, inactivation of interferingcomponents, the addition of reagents, lysing, etc. If such methods ofpretreatment are employed with respect to the sample, such pretreatmentmethods are typically such that the nucleic acid(s) of interest remainin the test sample, sometimes at a concentration proportional to that inan untreated test sample (e.g., namely, a sample that is not subjectedto any such pretreatment method(s)). Such “treated” or “processed”samples are still considered to be biological “test” samples withrespect to the methods described herein.

The term “qualified sample” or “unaffected sample” herein refers to asample comprising a mixture of nucleic acids that are present in a knowncopy number to which the nucleic acids in a test sample are to becompared, and it is a sample that is normal, i.e., not aneuploid, forthe sequence of interest. In some embodiments, qualified samples areused as unaffected training samples of a training set to derive sequencemasks or sequence profiles. In certain embodiments, qualified samplesare used for identifying one or more normalizing chromosomes or segmentsfor a chromosome under consideration. For example, qualified samples maybe used for identifying a normalizing chromosome for chromosome 21. Insuch case, the qualified sample is a sample that is not a trisomy 21sample. Another example involves using only females as qualifyingsamples for chromosome X. Qualified samples may also be employed forother purposes such as determining thresholds for calling affectedsamples, identifying thresholds for defining mask regions on a referencesequence, determining expected coverage quantities for different regionsof a genome, and the like.

The term “patient sample” herein refers to a biological sample obtainedfrom a patient, i.e., a recipient of medical attention, care ortreatment. The patient sample can be any of the samples describedherein. In certain embodiments, the patient sample is obtained bynon-invasive procedures, e.g., peripheral blood sample or a stoolsample. The methods described herein need not be limited to humans.Thus, various veterinary applications are contemplated in which case thepatient sample may be a sample from a non-human mammal (e.g., a feline,a porcine, an equine, a bovine, and the like).

The term “mixed sample” herein refers to a sample containing a mixtureof nucleic acids, which are derived from different genomes.

The term “maternal sample” herein refers to a biological sample obtainedfrom a pregnant subject, e.g., a woman or female of another species thatis pregnant.

The term “biological fluid” herein refers to a liquid in or from abiological source and includes, for example, blood, serum, plasma,sputum, lavage fluid, cerebrospinal fluid, urine, semen, sweat, tears,saliva, and the like. As used herein, the terms “blood,” “plasma” and“serum” expressly encompass fractions or processed portions thereof.Similarly, where a sample is taken from a biopsy, swab, smear, etc., the“sample” expressly encompasses a processed fraction or portion derivedfrom the biopsy, swab, smear, etc.

The terms “maternal nucleic acids” and “fetal nucleic acids” hereinrefer to the nucleic acids of a pregnant female subject and the nucleicacids of the fetus being carried by the pregnant female, respectively.

The term “sequence of interest” herein refers to a nucleic acid sequencethat is associated with a difference in sequence representation inhealthy versus diseased individuals. A sequence of interest can be asequence on a chromosome that is misrepresented, i.e., over- orunder-represented, in a disease or genetic condition. A sequence ofinterest may be a portion of a chromosome, i.e., chromosome segment, ora whole chromosome. For example, a sequence of interest can be achromosome that is over-represented in an aneuploidy condition, or agene encoding a tumor-suppressor that is under-represented in a cancer.Sequences of interest include sequences that are over- orunder-represented in the total population, or a subpopulation of cellsof a subject. A “qualified sequence of interest” is a sequence ofinterest in a qualified sample. A “test sequence of interest” is asequence of interest in a test sample.

The term “normalizing sequence” herein refers to a sequence that is usedto normalize the number of sequence tags mapped to a sequence ofinterest associated with the normalizing sequence. In some embodiments,a normalizing sequence comprises a robust chromosome. A “robustchromosome” is one that is unlikely to be aneuploid. In some casesinvolving the human chromosome, a robust chromosome is any chromosomeother than the X chromosome, Y chromosome, chromosome 13, chromosome 18,and chromosome 21. In some embodiments, the normalizing sequencedisplays a variability in the number of sequence tags that are mapped toit among samples and sequencing runs that approximates the variabilityof the sequence of interest for which it is used as a normalizingparameter. The normalizing sequence can differentiate an affected samplefrom one or more unaffected samples. In some implementations, thenormalizing sequence best or effectively differentiates, when comparedto other potential normalizing sequences such as other chromosomes, anaffected sample from one or more unaffected samples. In someembodiments, the variability of the normalizing sequence is calculatedas the variability in the chromosome dose for the sequence of interestacross samples and sequencing runs. In some embodiments, normalizingsequences are identified in a set of unaffected samples.

A “normalizing chromosome,” “normalizing denominator chromosome,” or“normalizing chromosome sequence” is an example of a “normalizingsequence.” A “normalizing chromosome sequence” can be composed of asingle chromosome or of a group of chromosomes. In some embodiments, anormalizing sequence comprises two or more robust chromosomes. Incertain embodiments, the robust chromosomes are all autosomalchromosomes other than chromosomes, X, Y, 13, 18, and 21. A “normalizingsegment” is another example of a “normalizing sequence.” A “normalizingsegment sequence” can be composed of a single segment of a chromosome orit can be composed of two or more segments of the same or of differentchromosomes. In certain embodiments, a normalizing sequence is intendedto normalize for variability such as process-related, interchromosomal(intra-run), and inter-sequencing (inter-run) variability.

The term “variability” herein refers to another characteristic of anormalizing chromosome that enables one to distinguish one or moreunaffected, i.e., normal, samples from one or more affected, i.e.,aneuploid, samples. The variability of a normalizing chromosome, whichis measured in a set of qualified samples, refers to the variability inthe number of sequence tags that are mapped to it that approximates thevariability in the number of sequence tags that are mapped to achromosome of interest for which it serves as a normalizing parameter.

The term “sequence tag density” herein refers to the number of sequencereads that are mapped to a reference genome sequence, e.g., the sequencetag density for chromosome 21 is the number of sequence reads generatedby the sequencing method that are mapped to chromosome 21 of thereference genome.

The term “sequence tag density ratio” herein refers to the ratio of thenumber of sequence tags that are mapped to a chromosome of the referencegenome, e.g., chromosome 21, to the length of the reference genomechromosome.

The term “sequence dose” herein refers to a parameter that relates thenumber of sequence tags identified for a sequence of interest and thenumber of sequence tags identified for the normalizing sequence. In somecases, the sequence dose is the ratio of the sequence tag coverage for asequence of interest to the sequence tag coverage for a normalizingsequence. In some cases, the sequence dose refers to a parameter thatrelates the sequence tag density of a sequence of interest to thesequence tag density of a normalizing sequence. A “test sequence dose”is a parameter that relates the sequence tag density of a sequence ofinterest, e.g., chromosome 21, to that of a normalizing sequence, e.g.,chromosome 9, determined in a test sample. Similarly, a “qualifiedsequence dose” is a parameter that relates the sequence tag density of asequence of interest to that of a normalizing sequence determined in aqualified sample.

The term “coverage” refers to the abundance of sequence tags mapped to adefined sequence. Coverage can be quantitatively indicated by sequencetag density (or count of sequence tags), sequence tag density ratio,normalized coverage amount, adjusted coverage values, etc.

The term “coverage quantity” is a modification of raw coverage and oftenrepresents the relative quantity of sequence tags (sometimes calledcounts) in a region of a genome such as a bin. A coverage quantity maybe obtained by normalizing, adjusting and/or correcting the raw coverageor count for a region of the genome. For example, a normalized coveragequantity for a region may be obtained by dividing the sequence tag countmapped to the region by the total number sequence tags mapped to theentire genome. Normalized coverage quantity allows comparison ofcoverage of a bin across different samples, which may have differentdepths of sequencing. It differs from sequence dose in that the latteris typically obtained by dividing by the tag count mapped to a subset ofthe entire genome. The subset is a normalizing segment or chromosome.Coverage quantities, whether or not normalized, may be corrected forglobal profile variation from region to region on the genome, G-Cfraction variations, outliers in robust chromosomes, etc.

The term “Next Generation Sequencing (NGS)” herein refers to sequencingmethods that allow for massively parallel sequencing of clonallyamplified molecules and of single nucleic acid molecules. Non-limitingexamples of NGS include sequencing-by-synthesis using reversible dyeterminators, and sequencing-by-ligation.

The term “parameter” herein refers to a numerical value thatcharacterizes a physical property. Frequently, a parameter numericallycharacterizes a quantitative data set and/or a numerical relationshipbetween quantitative data sets. For example, a ratio (or function of aratio) between the number of sequence tags mapped to a chromosome andthe length of the chromosome to which the tags are mapped, is aparameter.

The terms “threshold value” and “qualified threshold value” herein referto any number that is used as a cutoff to characterize a sample such asa test sample containing a nucleic acid from an organism suspected ofhaving a medical condition. The threshold may be compared to a parametervalue to determine whether a sample giving rise to such parameter valuesuggests that the organism has the medical condition. In certainembodiments, a qualified threshold value is calculated using aqualifying data set and serves as a limit of diagnosis of a copy numbervariation, e.g., an aneuploidy, in an organism. If a threshold isexceeded by results obtained from methods disclosed herein, a subjectcan be diagnosed with a copy number variation, e.g., trisomy 21.Appropriate threshold values for the methods described herein can beidentified by analyzing normalized values (e.g. chromosome doses, NCVsor NSVs) calculated for a training set of samples. Threshold values canbe identified using qualified (i.e., unaffected) samples in a trainingset which comprises both qualified (i.e., unaffected) samples andaffected samples. The samples in the training set known to havechromosomal aneuploidies (i.e., the affected samples) can be used toconfirm that the chosen thresholds are useful in differentiatingaffected from unaffected samples in a test set (see the Examplesherein). The choice of a threshold is dependent on the level ofconfidence that the user wishes to have to make the classification. Insome embodiments, the training set used to identify appropriatethreshold values comprises at least 10, at least 20, at least 30, atleast 40, at least 50, at least 60, at least 70, at least 80, at least90, at least 100, at least 200, at least 300, at least 400, at least500, at least 600, at least 700, at least 800, at least 900, at least1000, at least 2000, at least 3000, at least 4000, or more qualifiedsamples. It may advantageous to use larger sets of qualified samples toimprove the diagnostic utility of the threshold values.

The term “read” refers to sequence data from a portion of a nucleic acidsample. Typically, though not necessarily, a read represents a shortsequence of contiguous base pairs in the sample. The read may berepresented symbolically by the base pair sequence (in ATCG) of thesample portion. It may be stored in a memory device and processed asappropriate to determine whether it matches a reference sequence ormeets other criteria. A read may be obtained directly from a sequencingapparatus or indirectly from stored sequence information concerning thesample. In some cases, a read is a DNA sequence of sufficient length(e.g., at least about 25 bp) that can be used to identify a largersequence or region, e.g., that can be aligned and specifically assignedto a chromosome or genomic region or gene.

The term “sequence tag” is herein used interchangeably with the term“mapped sequence tag” to refer to a sequence read that has beenspecifically assigned, i.e., mapped, to a larger sequence, e.g., areference genome, by alignment. Mapped sequence tags are uniquely mappedto a reference genome, i.e., they are assigned to a single location tothe reference genome. Unless otherwise specified, tags that map to thesame sequence on a reference sequence are counted once. Tags may beprovided as data structures or other assemblages of data. In certainembodiments, a tag contains a read sequence and associated informationfor that read such as the location of the sequence in the genome, e.g.,the position on a chromosome. In certain embodiments, the location isspecified for a positive strand orientation. A tag may be defined toprovide a limit amount of mismatch in aligning to a reference genome. Insome embodiments, tags that can be mapped to more than one location on areference genome, i.e., tags that do not map uniquely, may not beincluded in the analysis.

The term “site” refers to a unique position (i.e. chromosome ID,chromosome position and orientation) on a reference genome. In someembodiments, a site may be a residue, a sequence tag, or a segment'sposition on a sequence. The term “locus” may be used to refer to thespecific location of a nucleic acid sequence or polymorphism on areference chromosome.

Normalized chromosome value (NCV) relates coverage of a test sample tocoverages of a set of training/qualified samples. In some embodiments,NCV is based on chromosome dose. In some embodiments, NCV relates to thedifference between the chromosome dose of a chromosome of interest in atest sample and the mean of the corresponding chromosome dose in a setof qualified samples as, and can be calculated as:

${NCV}_{ij} = \frac{x_{ij} - {\hat{\mu}}_{j}}{{\hat{\sigma}}_{j}}$where {circumflex over (μ)}_(j) and {circumflex over (σ)}_(j) are theestimated mean and standard deviation, respectively, for the j-thchromosome dose in a set of qualified samples, and x_(ij) is theobserved j-th chromosome ratio (dose) for test sample i.

In some embodiments, NCV can be calculated “on the fly” by relating thechromosome dose of a chromosome of interest in a test sample to themedian of the corresponding chromosome dose in multiplexed samplessequenced on the same flow cells as:

${NCV}_{ij} = \frac{x_{ij} - M_{j}}{{\hat{\sigma}}_{j}}$where M_(j) is the estimated median for the j-th chromosome dose in aset of multiplexed samples sequenced on the same flow cell; {circumflexover (σ)}_(j) is the standard deviation for the j-th chromosome dose inone or more sets of multiplexed samples sequenced on one or more flowcells, and x_(i) is the observed j-th chromosome dose for test sample i.In this embodiment, test sample i is one of the multiplexed samplessequenced on the same flow cell from which M_(j) is determined.

For example, for chromosome of interest 21 in test sample A, which issequenced as one of 64 multiplexed samples on one flow cell, the NCV forchromosome 21 in test sample A is calculated as the dose of chromosome21 in sample A minus the median of the dose for chromosome 21 determinedin the 64 multiplexed samples, divided by the standard deviation of thedose for chromosome 21 determined for the 64 multiplexed samples on flowcell 1, or of additional flow cells e.g. 20.

As used herein, the terms “aligned,” “alignment,” or “aligning” refer tothe process of comparing a read or tag to a reference sequence andthereby determining whether the reference sequence contains the readsequence or tag sequence. If the reference sequence contains the read,the read may be mapped to the reference sequence or, in certainembodiments, to a particular location in the reference sequence. In somecases, alignment simply tells whether or not a read is a member of aparticular reference sequence (i.e., whether the read is present orabsent in the reference sequence). For example, the alignment of a readto the reference sequence for human chromosome 13 will tell whether theread is present in the reference sequence for chromosome 13. A tool thatprovides this information may be called a set membership tester. In somecases, an alignment additionally indicates a location in the referencesequence where the read or tag maps to. For example, if the referencesequence is the whole human genome sequence, an alignment may indicatethat a read is present on chromosome 13, and may further indicate thatthe read is on a particular strand and/or site of chromosome 13.

Aligned reads or tags are one or more sequences that are identified as amatch in terms of the order of their nucleic acid molecules to a knownsequence from a reference genome. Alignment can be done manually,although it is typically implemented by a computer algorithm, as itwould be impossible to align reads in a reasonable time period forimplementing the methods disclosed herein. One example of an algorithmfrom aligning sequences is the Efficient Local Alignment of NucleotideData (ELAND) computer program distributed as part of the IlluminaGenomics Analysis pipeline. Alternatively, a Bloom filter or similar setmembership tester may be employed to align reads to reference genomes.See U.S. Patent Application No. 61/552,374 filed Oct. 27, 2011 which isincorporated herein by reference in its entirety. The matching of asequence read in aligning can be a 100% sequence match or less than 100%(non-perfect match).

The term “mapping” used herein refers to specifically assigning asequence read to a larger sequence, e.g., a reference genome, byalignment.

As used herein, the term “reference genome” or “reference sequence”refers to any particular known genome sequence, whether partial orcomplete, of any organism or virus which may be used to referenceidentified sequences from a subject. For example, a reference genomeused for human subjects as well as many other organisms is found at theNational Center for Biotechnology Information at ncbi.nlm.nih.gov. A“genome” refers to the complete genetic information of an organism orvirus, expressed in nucleic acid sequences.

In various embodiments, the reference sequence is significantly largerthan the reads that are aligned to it. For example, it may be at leastabout 100 times larger, or at least about 1000 times larger, or at leastabout 10,000 times larger, or at least about 105 times larger, or atleast about 106 times larger, or at least about 107 times larger.

In one example, the reference sequence is that of a full length humangenome. Such sequences may be referred to as genomic referencesequences. In another example, the reference sequence is limited to aspecific human chromosome such as chromosome 13. In some embodiments, areference Y chromosome is the Y chromosome sequence from human genomeversion hg19. Such sequences may be referred to as chromosome referencesequences. Other examples of reference sequences include genomes ofother species, as well as chromosomes, sub-chromosomal regions (such asstrands), etc., of any species.

In various embodiments, the reference sequence is a consensus sequenceor other combination derived from multiple individuals. However, incertain applications, the reference sequence may be taken from aparticular individual.

The term “clinically-relevant sequence” herein refers to a nucleic acidsequence that is known or is suspected to be associated or implicatedwith a genetic or disease condition. Determining the absence or presenceof a clinically-relevant sequence can be useful in determining adiagnosis or confirming a diagnosis of a medical condition, or providinga prognosis for the development of a disease.

The term “derived” when used in the context of a nucleic acid or amixture of nucleic acids, herein refers to the means whereby the nucleicacid(s) are obtained from the source from which they originate. Forexample, in one embodiment, a mixture of nucleic acids that is derivedfrom two different genomes means that the nucleic acids, e.g., cfDNA,were naturally released by cells through naturally occurring processessuch as necrosis or apoptosis. In another embodiment, a mixture ofnucleic acids that is derived from two different genomes means that thenucleic acids were extracted from two different types of cells from asubject.

The term “based on” when used in the context of obtaining a specificquantitative value, herein refers to using another quantity as input tocalculate the specific quantitative value as an output.

As used herein, the term “corresponding to” sometimes refers to anucleic acid sequence, e.g., a gene or a chromosome, that is present inthe genome of different subjects, and which does not necessarily havethe same sequence in all genomes, but serves to provide the identityrather than the genetic information of a sequence of interest, e.g., agene or chromosome.

As used herein, the term “fetal fraction” refers to the fraction offetal nucleic acids present in a sample comprising fetal and maternalnucleic acid. Fetal fraction is often used to characterize the cfDNA ina mother's blood.

As used herein the term “chromosome” refers to the heredity-bearing genecarrier of a living cell, which is derived from chromatin strandscomprising DNA and protein components (especially histones). Theconventional internationally recognized individual human genomechromosome numbering system is employed herein.

The term “subject” herein refers to a human subject as well as anon-human subject such as a mammal, an invertebrate, a vertebrate, afungus, a yeast, a bacterium, and a virus. Although the examples hereinconcern humans and the language is primarily directed to human concerns,the concepts disclosed herein are applicable to genomes from any plantor animal, and are useful in the fields of veterinary medicine, animalsciences, research laboratories and such.

The term “condition” herein refers to “medical condition” as a broadterm that includes all diseases and disorders, but can include[injuries] and normal health situations, such as pregnancy, that mightaffect a person's health, benefit from medical assistance, or haveimplications for medical treatments.

The term “complete” when used in reference to a chromosomal aneuploidyherein refers to a gain or loss of an entire chromosome.

The term “partial” when used in reference to a chromosomal aneuploidyherein refers to a gain or loss of a portion, i.e., segment, of achromosome.

The term “mosaic” herein refers to denote the presence of twopopulations of cells with different karyotypes in one individual who hasdeveloped from a single fertilized egg. Mosaicism may result from amutation during development which is propagated to only a subset of theadult cells.

The term “non-mosaic” herein refers to an organism, e.g., a human fetus,composed of cells of one karyotype.

The term “sensitivity” as used herein is equal to the number of truepositives divided by the sum of true positives and false negatives.

The term “specificity” as used herein is equal to the number of truenegatives divided by the sum of true negatives and false positives.

The term “enrich” herein refers to the process of separating orselectively amplifying a subset of nucleic acids contained in a sample.Enrichment includes specific enrichment that targets specific sequences,e.g., polymorphic sequences, and non-specific enrichment that amplifiesthe whole genome of the DNA fragments of the sample.

The term “primer,” as used herein refers to an isolated oligonucleotidethat is capable of acting as a point of initiation of synthesis whenplaced under conditions inductive to synthesis of an extension product(e.g., the conditions include nucleotides, an inducing agent such as DNApolymerase, and a suitable temperature and pH). The primer is preferablysingle stranded for maximum efficiency in amplification, but mayalternatively be double stranded. If double stranded, the primer isfirst treated to separate its strands before being used to prepareextension products. Preferably, the primer is anoligodeoxyribonucleotide. The primer must be sufficiently long to primethe synthesis of extension products in the presence of the inducingagent. The exact lengths of the primers will depend on many factors,including temperature, source of primer, use of the method, and theparameters used for primer design.

The term “polymorphism” or “genetic polymorphism” is used herein inreference to the occurrence in the same population of two or morealleles at one genetic locus. Various forms of polymorphism includesingle nucleotide polymorphisms, tandem repeats, micro-deletions,insertions, indels, and other polymorphisms.

The term “training set” and “training sequences” are usedinterchangeably herein and refer to a set of genetic sequences obtainedto derive quantitative estimates for one or more sequences of interest.Examples of the estimates derivable include the probability distributionof quantities or properties of the sequences of interest (e.g., mean andstandard deviation of allele counts or sequence coverage), relationsamong sequences of interest (e.g., differences of coverages).

Introduction

In various embodiments, the method can determine one or more sequencesof interest in a test sample that comprises a mixture of single-genomecellular DNA and mother-and-fetus cfDNA. In some embodiments, thesingle-genome cellular DNA is maternal DNA; in other embodiments, thesingle-genome cellular DNA is fetus DNA. In particular embodiments,single-genome cellular nucleic acid material (e.g. genomic DNA) isobtained from fetal cells that are isolated from the blood of a pregnantfemale, and also mother-and-fetus cell free nucleic acid material (e.g.genomic cfDNA) is obtained from blood of the same female. Typically,fetal cells are in relatively low abundance in maternal blood, such thatrelatively low quantities of fetal nucleic acids are available from theamount of blood that is comfortably drawn from a pregnant mother.However, fetal cells provide a convenient ‘package’ for separating fetalnucleic acids from the relatively high background of maternal nucleicacid that is found in the blood of a pregnant female. Conversely,relatively large amounts of cell-free fetal nucleic acid can be obtainedfrom a standard blood draw, albeit along with a relatively highbackground of maternal nucleic acid material. The present disclosureprovides methods and compositions that exploit the relatively high yieldof fetal nucleic acids obtained from cell-free fractions and therelatively pure fetal nucleic acid obtained from fetal cells to providea combined method with emergent benefits that are greater than thebenefits provided by the methods when carried out individually. By wayof more specific example, nucleic acids, once obtained from cellular andcell-free fractions, respectively, can be re-combined for analyticalsteps in a method set forth herein. The relatively abundant nucleicacids in the mixed mother-and-fetus fraction can be detected robustlyand with high technical/statistical confidence in this combinedanalytical process and the relatively pure fetal nucleic acid can beused as an internal reference to distinguish characteristics of thefetal nucleic acid that may otherwise have been difficult to distinguishin the mixed mother-and-fetus fraction.

Methods, apparatus, and systems are disclosed herein for determining atleast one sequence of interest of a fetus of a pregnant mother. In someembodiments involving analyses of mother-only cellular DNA, thedisclosed embodiments permit determining the zygosity case (e.g.,homozygous in a minor allele) of fetal DNA obtained from cfDNA. In someembodiments, the method can determine one or more sequences of interestin a test sample that contains a mixture of mother-only cellular DNA andmother-and-fetus cfDNA. The sequences of interest determined by themethods and apparatus disclosed herein may include one or more of thefollowing: single nucleotide polymorphisms, tandem repeats,micro-deletions, insertions, and other polymorphisms. In someembodiments, the method may determine copy number of sequence ofinterests, including gains or losses of entire chromosomes, alterationsinvolving very large chromosomal segments that are microscopicallyvisible, and an abundance of sub-microscopic copy number variation ofDNA segments ranging from single nucleotide, to kilobases (kb), tomegabases (Mb) in size.

In some embodiments, methods are provided for determining whether thefetus has a genetic condition, or more specifically, for determiningwhether the fetus is homozygous in a disease causing allele when themother is heterozygous of the same allele. When the mother is a carrierfor a recessive genetic condition, the fetus has a 25% chance ofdeveloping the genetic condition if the father is also a carrier. Insuch case, the mother is heterozygous of the gene for the condition,having one causative allele and one normal allele; the fetus ishomozygous of the gene, having two copies of the causative allele. It isdesirable to determine if the fetus has inherited particular alleles(e.g. disease causing alleles) from both parents and to do so in anon-invasive manner using maternal plasma cfDNA. However, it isdifficult to differentiate if the fetus is homozygous or heterozygouswhen the mother is heterozygous using conventional method ofnon-invasive prenatal diagnosis (NIPD) because only those SNPs for whichthe mother is homozygous and the fetus is heterozygous were considered“informative SNPs.” SNPs where the mother may be heterozygous and thefetus is homozygous cannot be used without understanding the inherent“noise” in the heterozygous calls for the mother.

By genotyping mother-only DNA, the current disclosure provides methodsto use the majority component of genetic materials that contributes tothe data from the mixed genome cfDNA. One can then evaluate multipleSNPs or other polymorphisms from the maternal cellular DNA to assist thezygosity call for a polymorphism of interest. This may be done with orwithout explicitly determining the fetal fraction in the cfDNA.

Processing Workflow Involving Analyses of Mother-Only Cellular DNA

To detect if a fetus of a carrier mother will manifest a recessivegenetic trait, one can determine if the fetus is homozygous for thedisease-causing alleles in a background where the mother isheterozygous. In some embodiments, one takes advantage of the fact that,in maternal blood, mother-only cellular DNA can be obtained from thewhite blood cells of the buffy coat and the mother-and-fetus mixed cfDNAcan be obtained from the plasma. Information from the mother-onlycellular DNA can be used to reduce the noise in the data, therebyhelping to differentiate the homozygous fetus case from heterozygousfetus case (when mother is heterozygous). In some embodiments, atargeted amplification and sequencing method can be used for thispurpose. To reduce processing biases and otherwise permit reliablecomparison of the cfDNA sequences and the cellular DNA sequences, thetwo DNA sources are processed similarly; for example, they are amplifiedand/or sequenced by multiplexing. In some embodiments, the cellular DNAand cfDNA are obtained from the same sample but then separated andindexed (or otherwise uniquely identified) and then pooled for locusspecific amplification, sequencing, and the like. In someimplementations, the separately indexed cellular DNA and cfDNA are madesimilar with regard to size and concentration prior to pooling formultiplexed amplification, sequencing, and other downstream processing.It will be understood that the methods exemplified herein for use withDNA can use other nucleic acids such as mRNA.

FIG. 1 shows a process flow of a method 100 for determining a sequenceof interest of a fetus. In some embodiments, the method 100 involvesobtaining mother-only cellular DNA of the pregnant mother (e.g. fromisolated maternal cells). See block 102. The mother-only cellular DNAincludes at least a sequence that maps to the sequence of interest. Insome embodiments, the sequence of interest includes polymorphicsequences of a disease related gene. In some embodiments, the sequenceof interest comprises a site of an allele associated with a disease. Insome embodiments, the sequence of interest comprises one or more of thefollowing: single nucleotide polymorphism, tandem repeat,micro-deletion, and insertion.

In some embodiments, the cellular DNA is obtained from the white bloodcells fractionated into the buffy coat of a blood sample from thepregnant mother. The cellular DNA may also be obtained from cells thatare lysed after removal from other tissues and fluid as describedhereinafter.

In some embodiments, the method also involves obtaining mother-and-fetusmixed cfDNA from the pregnant mother. See block 104. The cfDNA includesat least one sequence that maps to the at least one sequence ofinterest. In some embodiments, the cfDNA is obtained from the plasma ofa blood sample from the mother. In some embodiments, the blood samplealso provides the buffy coat as the source of the cellular DNA.

In some embodiments, the method employs an indicator of the source ofDNA as being from the mother-only cellular DNA or from the cfDNA. Seeblock 106. In some embodiments, this indicator is provided by preparinga first sequencing library of the mother-only cellular DNA and a secondsequencing library of the cfDNA. In preparing sequence libraries, themethod may involve incorporating indexes to one or both of saidsequencing libraries. When indexes are incorporated into both libraries,the indexes incorporated to the first library will differ from theindexes incorporated to the second library. Typically the indexescontain unique sequences (e.g., bar codes) that are identifiable indownstream sequencing steps, thereby providing an indicator of thesource of the nucleic acids.

In some embodiments, the method proceeds by combining at least a portionof the mother-only cellular DNA and a least a portion of the cfDNA toprovide a sample of combined cellular DNA and cfDNA. See block 108. Insome embodiments, the cellular DNA and cfDNA are of similar quantityand/or concentration. However, the methods can accommodate a relativeskew in quantity, for example where the amount of cellular DNA is lessthan the amount of cfDNA. In some embodiments, the method involvesfurther processing the combined sample to prepare or modify sequencinglibraries. In some embodiments, this involves incorporating sequencingadaptors (e.g., paired end primers) for massively parallel sequencing.

In some embodiments, the method then proceeds with sequencing at least aportion of the sample of combined cellular and cfDNA to provide aplurality of sequence reads. See block 110. The sequence reads are thenmapped to a reference sequence containing the sequence of interest orcompared to the sequence of interest, thereby providing sequence tagsmapped to the sequence of interest. The sequence of interest mayidentify the presence of an allele. In many embodiments, the sample hasbeen selectively enriched for the sequence of interest.

In some embodiments, the sample has not been selectively enriched forthe sequence of interest before sequencing, wherein the sample may beamplified by whole genome amplification. In these embodiments, thesequence reads are aligned to a reference genome comprising a sequenceof interest (e.g., chromosome, chromosome segment) that are typicallylonger than in the embodiment with selective enrichment targetingshorter sequences of interest (e.g., SNPs, STRs, and sequences of up tokb in size). The sequence reads mapping to the sequence of interestprovide sequence tags for the sequence of interest, which can be used todetermine a genetic condition related to the sequence of interest.

In some embodiments, the method applies massively parallel sequencing.Various sequencing techniques may be used, including but not limited to,sequencing by synthesis and sequencing by ligation. In some embodiments,sequencing by synthesis uses reversible dye terminators. In someembodiments, single molecule sequencing is used.

In some embodiments, the method further involves analyzing a pluralityof sequence tags to determine at least one sequence of interest. Seeblock 112. At least a portion of the plurality of sequence tags map tothe at least one sequence of interest. In some embodiments, the methoddetermines the presence or abundance of sequence tags mapping to thesequence of interest. Particularly, the method may determine therelative amounts of two alleles in each of the cfDNA and cellular DNA.In some embodiments, the method may detect that the fetus has a geneticdisorder by determining that the fetus is homozygous of a diseasecausing allele of a disease related gene wherein the mother isheterozygous of the allele. In some embodiments, the at least onesequence of interest comprises a site of an allele associated with adisease. In some embodiments, the method further includes determining ifthe fetus is homozygous or heterozygous for the disease associatedallele.

In some embodiments, the method starts with cellular DNA and cfDNA inseparate reaction environments, e.g., test tubes. The method involvesenriching wild-type and mutant regions using probes that target bothalleles of disease related gene(s) and have different indices forcellular DNA and cfDNA, the indices are incorporated into the targetedsequences in the separate reaction environment. The method furtherinvolves mixing the cellular DNA and cfDNA with enriched targetedregions and amplifying the DNA using universal PCR primers. Theamplified product will be sequencing-ready targeted libraries of bothcellular DNA for the mother and cfDNA for the mother and fetus. Thesequencing results may then be used to determine a sequence of interestfor the fetus. The method may determine the zygosity of the fetus and/orfetal fraction of the cfDNA.

In some embodiments, the method further involves determining thesequence of interest from the cfDNA and the cellular DNA. It alsoinvolves determining at least one or a plurality of training sequencefrom the cfDNA and the cellular DNA. The training sequences may be anysequences with ascertainable zygosity, such as sequences in the sexchromosomes or other sequences having polymorphisms whose zygosity casescorrelate with observable phenotypes. Some embodiments further determinea zygosity case of the sequence of interest for the fetus. In someembodiments, the zygosity case is determined by comparing the relativeamounts of two alleles in the cellular DNA and cfDNA mapping to thesequence of interest and mapping to training sequences. In someembodiments, the zygosity case for the fetus is determined by comparingthe relative amounts of DNA mapping to two or more alleles.

FIG. 2 shows a workflow of a method for determining conditions relatedto a sequence of interest for a fetus as in some embodiments of thedisclosure. The method involves obtaining a blood sample from thepregnant mother carrying the fetus. See block 202. In some embodiments,it is assumed that the mother is the genetic mother of the fetus. Thisassumption determines the possible zygosity cases of the fetus given thezygosity of the mother. For instance, if the mother is homozygous ofallele a (i.e., a/A) for a biallelic gene having allele a and allele A,the genetic fetus of the mother can only be homozygous a/a, orheterozygous a/A. But for a homozygous (a/a) surrogate mother who is notthe genetic mother of the fetus, the fetus may be homozygous a/a, A/A,or heterozygous a/A. The analysis of described herein elsewhere canchange its zygosity assumption depending on whether or not the mother isthe genetic mother of the fetus.

In some embodiments, the method separates a buffy coat component and aplasma component of the blood sample. See block 204. Separation may beaccomplished by fractionation, centrifugation, etc. as describedhereinafter. In some embodiments, the method involves obtainingmother-only cellular DNA from the buffy coat. The cellular DNA maps tothe sequence of interest. See block 206. In some embodiments, the methodalso involves obtaining mother-and-fetus mixed cfDNA from the plasma,where the cfDNA maps to the sequence of interest. See block 208. In someembodiments, the method provides an indicator of the source of DNA asbeing from the cellular DNA or the cfDNA. See block 210. In someembodiments, the indicator is provided by two or more different indexsequences for the cellular DNA or the cfDNA. In some embodiments, themethod involves combining the cellular DNA and the cfDNA. See block 212.Then the method involves sequencing the combined cellular and cfDNA toprovide a plurality of sequence tags. See block 214. In someembodiments, the method involves analyzing the plurality of sequencetags to determine the sequence of interest. See block 216.

Example Workflow Using Two Primers when Incorporating Indexes

Plasma cfDNA from a pregnant woman is a mixture comprised mostly ofmother DNA with a certain fraction of fetal DNA. An accurate and precisedetermination of the % fetal DNA in maternal plasma cfDNA is desirablein non-invasive prenatal testing, especially for samples with low fetalfractions. One commonly used method for determining fetal fractioninterrogates a plurality of high heterozygocity SNPs and studies allelicfrequency differences between mother and fetus. Data analysis can bechallenging when one studies SNPs only in mixed maternal plasma cfDNA,since “true” genotype of the mother alone or the fetus alone is notknown.

In NIPD, it is difficult to get pure fetal DNA. However, determinationof fetal fraction in maternal plasma cfDNA samples can be simplified ifone is able to account for known mother-only SNP allelic frequenciesfrom the mixture, since the mother DNA constitutes the majority of thecfDNA. Mother-only cellular DNA can be obtained from the buffy coat ofthe patient blood sample. In order to correctly account for themother-only SNP data from the mixed plasma cfDNA SNP data, thebiochemical steps to which the two DNAs (buffy coat and plasma) aresubjected can be carried out together in the same processing system(s)at the same time, to reduce the risk of introducing process bias.

FIG. 3 shows a diagram of a workflow according to some embodiments ofthe invention, wherein the incorporation of index sequences occur withtwo primers introducing two adaptor segments. The figure presents aworkflow where the mother and the plasma mixture DNAs are indexedindependently during an early PCR step that also interrogates aplurality of SNP-sites in a multiplexed manner. The indexing at thisstage is called “source index” since it differentiates betweenmother-only DNA (e.g. maternal cellular DNA) and the mixedmaternal-fetal cfDNA within the same patient sample.

As shown in FIG. 3, the source index 1 sequence is incorporated into themother-only cellular DNA, and the source index 2 is incorporated intothe cfDNA, when the cellular DNA and the cfDNA are processed separately.In this indexing stage, two primers are applied, both targeting the SNPof interest, which is shown as a dot at the center of the two sequences.The primers to the left of the SNP include a sequence index as well asabout one half of a paired end adaptor (e.g., the P5 adaptor for theIllumina sequencing platform). Only about half of a sequencing adaptoris introduced by the primer to avoid a long overhang, which coulddehybridize too easily in some conditions. The remaining portion of theadaptor is introduced at later processing stages. The primers to theright of the SNP in the depicted example include about one half ofsecond paired end adaptor (e.g., a P7 adaptor for the Illuminasequencing platform). The P5 and P7 adaptors allow library fragments toanneal to their complementary oligos on the flowcell surface of theIllumina sequencer, which adaptors facilitate performance of bridgeamplification (e.g. clustering).

One point to consider at the indexing stage is to keep the number ofamplification cycles minimal, so as to minimize bias between the twosamples. In the depicted workflow, amplicons are independently cleanedusing Solid Phase Reversible Immobilization (SPRI), then eluted in equalvolume and pooled. The amplicons are then mixed together and subjectedto universal PCR at the same time in a single tube. The universal PCRintroduces a second index at the P7 end, which second index can be usedto multiplex patient samples during sequencing. During the universalPCR, P5 and P7 adaptors are extended to full length. By the end of PCR,library fragments are prepared to include all sequences necessary formultiplexed sequencing. Library fragments are SPRI-cleaned and ready forsequencing.

Since each kind of DNA from the same patient sample is source-indexeduniquely, the downstream data from a given patient can be identified asarising from mother-only DNA (e.g. maternal cellular DNA) and from mixedmother-fetus DNA (e.g. cfDNA). The mother data can then be subtractedfrom the mixed DNA data. The resulting data provides a means todetermine the zygosity and the fetal fraction of the SNPs of interest,which means is associated with lower noise and higher discriminationpower between different zygosity cases.

In some prior methods, only those SNPs for which the mother ishomozygous and the fetus is heterozygous constitute “informative SNPs.”SNPs where the mother may be heterozygous and the fetus is homozygousare not easily used without understanding the inherent “noise” in theheterozygous calls for the mother. By genotyping mother-only DNA, themajority component of that contributes to the data from the mixed plasmacfDNA is now known using the method of the current disclosure. One canthen use all SNPs where there is a difference between mother and fetusto calculate fetal fraction.

It will be understood, that biochemical steps and analytical stepsexemplified above for processing maternal only DNA with mixedmaternal-fetal DNA from a patient sample can be similarly carried outusing fetal only DNA (e.g. fetal cellular DNA) with the mixedmaternal-fetal DNA. For example, the method can include a step ofisolating fetal cells from maternal blood using, for example, antibodiesto fetal cell surface antigens. The antibodies can be attached to solidphase to allow separation and/or the antibodies can be used to detectfetal cells separated using known cell isolation techniques. Exemplarytechniques that can be used to isolate fetal cells from maternal bloodare set forth in U.S. Pat. Nos. 8,071,395 and 8,168,389, each of whichis incorporated herein by reference. The methods can also be carried outwith nucleic acids other than DNA, for example, mRNA.

Example Workflow Using One Primer when Incorporating Indexes

FIG. 4 presents a workflow similar to that of example 2, but it usesonly one primer and introduces only one sequencing adaptor (P5) whenincorporating indexes. The workflow introduces a second adaptor (P7)after two samples have been indexed and combined. The mother DNA and theplasma mixture DNA are indexed independently with a “source-index” at afirst step extension. A single-primer based cyclic extension reaction isdone in two separate tubes using multiplexed locus-specific extensionoligos (F-LSO) which introduce the source-index and ½ of the P5 adapter.A separate locus-specific extension oligo is used for each SNP (trainingand otherwise) under consideration. After a relatively small numbersingle extension cycles, the samples are pooled and subjected to PCRwith the reverse LSO (R-LSO) which also introduces ½ of the P7 adapterand the remainder of the P5 adapter. A final universal PCR introducesall of the necessary sequencing adapters along with a patient-specificindex at the P7 end. The workflow is otherwise comparable to that ofExample 2. Again, the methods can be carried out using fetal-only DNAinstead of maternal-only DNA. Alternatively or additionally, othernucleic acids, such as mRNA can be used.

Example Workflow Using Ligation to Incorporate Index

FIGS. 5A and 5B describe a workflow where the maternal only and theplasma mixture DNAs are indexed independently, mixed together and thensubjected to the multiplexed SNP-interrogation at the same time in asingle tube. The workflow uses ligation to incorporate two copies of thesame index sequence into two ends of a fragment, which index sequencecopies on two ends of the fragment then self-hybridize and form astem-loop structure when the single strand fragment is denatured.

FIG. 5A shows the indexing operation that ligates a double stranded P7adaptor and an index to the A-tailed fragments. The cellular DNAobtained from the buffy coat and the cfDNA from the plasma are indexedseparately in separate tubes. The cellular DNA is enzymaticallyfragmented and SPRI-size selected to have fragments of about 170 bpbefore index ligation. The ligated fragments are then SPRI-cleaned andquantified.

FIG. 5B shows the indexed fragments are combined and further processedto selectively enrich and amplify the sequence of interest. The workflowproceeds by mixing approximately equal concentration and amount ofindexed cellular DNA and cfDNA. After denaturing and renaturing, DNAfragments form stem-loop structures upon renaturing. Then the processapplies two primers: a P7 primer and LSO primer that includes one halfof P5 sequence. These primers are used in limited cycles of multiplexPCR, to pull down multiple locus-specific polymorphisms, and incorporatethe P5 primer to the DNA fragments. Then universal PCR is performed toamplify the library fragments and to complete the extension of the P7and P5 adaptors.

At the end of the universal PCR, all necessary sequences are availablefor multiplexed sequencing. Since each kind of DNA from the same patientsample is source-indexed uniquely, the downstream data from a givenpatient can be identified as arising as from mother-only and from themixed DNA populations. The maternal only data can then be subtractedfrom the mixed DNA data. The methods exemplified in this workflow can becarried out using fetal-only DNA instead of maternal-only DNA.Alternatively or additionally, other nucleic acids, such as mRNA can beused.

Sequencing Library Preparation Involving Mother-Only Cellular DNA

In some embodiments, the method for determining a sequence of interestfor a fetus uses a step of incorporating indexes into sequencefragments. Indexes are identifiable during downstream processing andanalyses, providing indicators to identify the source of the geneticmaterials. In some embodiments, the indexes indicate whether thematerial is derived from a cellular nucleic acid fraction or cell-freenucleic acid fraction. In some embodiments, the indexes are incorporatedinto the sequences by hybridizing and extending primer sequencescomprising the indexes. In some embodiments, the primer sequences alsocomprise locus-specific extension oligonucleotides. This allows theprimer to target sequence of interest, thereby allowing selectiveenrichment of the sequence of interest before further amplificationand/or sequencing. In some embodiments, the locus-specific extensionoligonucleotides are selective to two or more alleles of a diseaserelated gene, thereby allowing the primer to target the two alleles ofthe disease related gene. In some embodiments, the primer sequences alsoinclude adaptors, or portions of adaptors, for next generationsequencing. In some embodiments, indexes are incorporated by ligationinstead of primer extension, which may involve ligating sequencescomprising the indexes. In some embodiments, the indexes may beincorporated ligating sequences that also contain sequencing adaptors,or portions thereof, for next generation sequencing. In someembodiments, indexes and/or adaptors are mediated by Tn5 transposase,and the sequencing libraries are transposon insertion libraries.

In some embodiments, the method also involves incorporating anindividual-specific index to the sequencing libraries. Theindividual-specific index indicates the identity of the pregnant motherthat provides the sample. This allows the pregnant mother's nucleicacids to be processed with other individuals' nucleic acids for parallelsequencing.

In some embodiments, the method involves starting with substantially thesame size and/or substantially the same concentration of cellular DNAand cfDNA in separate reaction environments, e.g., testing tubes. Invarious embodiments, the sizes and/or concentrations for two reactionsare within about 5%, 10%, 15%, 20%, 30%, 40%, or 50% of one another. Themethod involves enriching wild-type and mutant regions using probes thattarget both alleles of disease related gene(s) and have differentindices for cellular DNA and cfDNA, the indices are incorporated intothe targeted sequences in the separate reaction environment. The methodfurther involves mixing the cellular DNA and cfDNA with enrichedtargeted regions and amplifying the DNA using universal PCR primers. Theamplified product will be sequencing-ready targeted libraries of bothcellular DNA for the mother and cfDNA for the mother and fetus. Thesequencing results may then be used to determine a sequence of interestfor the fetus. The method may determine the zygosity of the fetus and/orfetal fraction of the cfDNA.

In some embodiments, the method further comprises amplifying a pluralityof training sequences before sequencing the combined sample. In someembodiments, the plurality of training sequences comprises more than 10,50, 60, 100, 500, 1000, or 5000 sequences. In some embodiments, some orall of the training sequences contain polymorphisms, e.g., onepolymorphism per training sequence. In some embodiments, the methodfurther comprises obtaining distribution statistics of sequence tagcounts for the training sequences. In some embodiments, the methodfurther involves using the distribution statistics to determine thezygosity of the sequence of interest for the fetus.

In some embodiments, the cellular DNA and cfDNA are also enriched fortraining sequences or sites. In some embodiments, more than about 10,50, 60, 100, 500, 1000, or 5000 SNP sites are used as training sites tounderstand distribution counts of SNPs sequence tags in the assay wherethe mother is heterozygous and the fetus is homozygous.

In some embodiments, the methods described herein can utilize nextgeneration sequencing technologies (NGS), that allow multiple samples tobe sequenced individually as genomic molecules (i.e., singleplexsequencing) or as pooled samples comprising indexed nucleic acids (e.g.,multiplex sequencing) on a single sequencing run. These methods cangenerate up to several hundred million reads of nucleic acid sequences.In various embodiments the sequences of genomic nucleic acids, and/or ofindexed genomic nucleic acids can be determined using, for example, theNext Generation Sequencing Technologies (NGS) described herein. Invarious embodiments analysis of the massive amount of sequence dataobtained using NGS can be performed using one or more processors asdescribed herein.

In certain embodiments the sequencing methods contemplated hereininvolve the preparation of sequencing libraries. In one illustrativeapproach, sequencing library preparation involves the production of arandom collection of adapter-modified DNA fragments (e.g.,polynucleotides) that are ready to be sequenced. Sequencing libraries ofpolynucleotides can be prepared from DNA or RNA, including equivalents,analogs of either DNA or cDNA, for example, DNA or cDNA that iscomplementary or copy DNA produced from an RNA template, by the actionof reverse transcriptase. The polynucleotides may originate indouble-stranded form (e.g., dsDNA such as genomic DNA fragments, cDNA,PCR amplification products, and the like) or, in certain embodiments,the polynucleotides may originated in single-stranded form (e.g., ssDNA,RNA, etc.) and have been converted to dsDNA form. By way ofillustration, in certain embodiments, single stranded mRNA molecules maybe copied into double-stranded cDNAs suitable for use in preparing asequencing library. The precise sequence of the primary polynucleotidemolecules is generally not material to the method of librarypreparation, and may be known or unknown. In one embodiment, thepolynucleotide molecules are DNA molecules. More particularly, incertain embodiments, the polynucleotide molecules represent the entiregenetic complement of an organism or substantially the entire geneticcomplement of an organism, and are genomic DNA molecules (e.g., cellularDNA, cell free DNA (cfDNA), etc.), that typically include both intronsequence and exon sequence (coding sequence), as well as non-codingregulatory sequences such as promoter and enhancer sequences. In certainembodiments, the primary polynucleotide molecules comprise human genomicDNA molecules, e.g., cfDNA molecules present in peripheral blood of apregnant subject.

Preparation of sequencing libraries for some NGS sequencing platforms isfacilitated by the use of polynucleotides comprising a specific range offragment sizes. Preparation of such libraries typically involves thefragmentation of large polynucleotides (e.g. cellular genomic DNA) toobtain polynucleotides in the desired size range.

Fragmentation can be achieved by any of a number of methods known tothose of skill in the art. For example, fragmentation can be achieved bymechanical means including, but not limited to nebulization, sonicationand hydroshear. However mechanical fragmentation typically cleaves theDNA backbone at C—O, P—O and C—C bonds resulting in a heterogeneous mixof blunt and 3′- and 5′-overhanging ends with broken C—O, P—O and/C—Cbonds (see, e.g., Alnemri and Liwack, J Biol. Chem 265:17323-17333[1990]; Richards and Boyer, J Mol Biol 11:327-240 [1965]) which may needto be repaired as they may lack the requisite 5′-phosphate for thesubsequent enzymatic reactions, e.g., ligation of sequencing adaptors,that are required for preparing DNA for sequencing.

In contrast, cfDNA, typically exists as fragments of less than about 300base pairs and consequently, fragmentation is not typically necessaryfor generating a sequencing library using cfDNA samples.

Typically, whether polynucleotides are forcibly fragmented (e.g.,fragmented in vitro), or naturally exist as fragments, they areconverted to blunt-ended DNA having 5′-phosphates and 3′-hydroxyl.Standard protocols, e.g., protocols for sequencing using, for example,the Illumina platform as described elsewhere herein, instruct users toend-repair sample DNA, to purify the end-repaired products prior todA-tailing, and to purify the dA-tailing products prior to theadaptor-ligating steps of the library preparation.

Solid Phase Reversible Immobilization (SPRI) beads are widely used forpurification of PCR amplified colonies in several DNA sequencingprotocols. SPRI beads are paramagnetic (magnetic only in a magneticfield) and this prevents them from clumping and falling out of solution.Each bead is made of polystyrene surrounded by a layer of magnetite,which is coated with carboxyl molecules, which reversibly bind DNA inthe presence of the “crowding agent” polyethylene glycol (PEG) and salt(20% PEG, 2.5M NaCl is the magic mix).

In some embodiments, the sequencing libraries are transposon insertionlibraries. Various embodiments of methods of sequence librarypreparation described herein obviate the need to perform one or more ofthe steps typically mandated by standard protocols to obtain a modifiedDNA product that can be sequenced by NGS. An abbreviated method (ABBmethod), a 1-step method, and a 2-step method are examples of methodsfor preparation of a sequencing library, which can be found in U.S.patent application Ser. No. 13/555,037 filed on Jul. 20, 2012, which isincorporated by reference by its entirety.

Processing Workflow Involving Analyses of Fetus-Only Cellular DNA

Overall Workflow Involving Analyses of Fetus-Only Cellular DNA

This section describes how biological samples from a pregnant mother isobtained to extract fetal cellular DNA and fetus-and-mother cfDNA, whichare then used to prepare libraries that provide DNA to deriveinformation for analysis of a sequence of interest. In some embodimentsthe sequence of interest includes a single nucleotide polymorphism thatis related to a medical condition or biological trait. In theembodiments that involve chromosomes or segments of chromosomes, themethods disclosed herein may be used to identify monosomies ortrisomies, e.g. trisomy 21 that causes Down Syndrome.

In some embodiments, fetal cellular DNA can be obtained from fetalnucleated red blood cells circulating in the maternal blood, andmother-and-fetus mixed cfDNA can be obtained from the plasma of thematernal blood. The two sources of DNA are then combined and furtherprocessed together to obtain two sequencing libraries having indexesidentifying the sources of the DNA. The sequencing information obtainedfrom the two libraries is used to determine a sequence of interest. Forinstance, in some embodiments, sequence information from the fetalcellular DNA can be used to validate a mosaicism call obtained fromcfDNA analysis. Additionally, the combination of sequence informationfrom both the fetal cellular DNA and the cfDNA may provide a higherconfidence interval and/or reduce noise in calls for copy numbervariation, fetal fraction, and/or fetal zygosity. For instance,information from the fetal cellular DNA can be used to reduce the noisein the data, thereby helping to differentiate a homozygous fetus from aheterozygous fetus case (when the mother is heterozygous).

In some embodiments, a targeted amplification and sequencing method canbe used. In other embodiments, whole genome amplification may be appliedbefore sequencing. To reduce processing biases and otherwise permitreliable comparison of the cell free nucleic acid sequences and thecellular nucleic acid sequences, the two nucleic acid samples areprocessed similarly in some embodiments. For example, they can besequenced in a mixture of the nucleic acids from both samples by amultiplexing technique. In some embodiments, cellular nucleic acids andcell free nucleic acids are obtained from the same sample but thenseparated and indexed (or otherwise uniquely identified) in theseparated fractions and then the fractions are pooled for amplification,sequencing, and the like. In some implementations, the fetal cellularnucleic acid fraction is enhanced before being combined withmother-and-fetus cell free nucleic acid fraction, such that theseparately indexed cellular nucleic acid and cell free nucleic acid aremade similar with regard to size and concentration prior to pooling forsequencing and other downstream processing.

FIG. 6 shows a process flow of a method 600 for determining a sequenceof interest of a fetus according to some embodiments of the disclosure.FIGS. 7-12 are specific implementations of various components of theprocess flow depicted in FIG. 6. In some embodiments, method 600involves obtaining cellular DNA from a maternal blood sample of apregnant mother. See block 602. In some embodiments, the cellular DNAincludes both maternal cellular DNA and fetal cellular DNA. In someembodiments, the fetal cellular DNA is isolated from maternal cellularDNA before further downstream processing. The fetal cellular DNAincludes at least a sequence that maps to the sequence of interest. Insome embodiments, the sequence of interest includes polymorphicsequences of a disease related gene. In some embodiments, the sequenceof interest comprises a site of an allele associated with a disease. Insome embodiments, the sequence of interest comprises one or more of thefollowing: single nucleotide polymorphism, tandem repeat, deletion,insertion, a chromosome or a segment of a chromosome.

In some embodiments, fetal cellular DNA is obtained from fetal nucleatedred blood cells (NRBCs) circulating in the maternal blood sample. Thefetal cellular DNA and the fetal NRBCs may be obtained from maternalperipheral blood as described herein. In some embodiments, the fetalNRBCs are obtained from an erythrocyte fraction of a maternal bloodsample. In some embodiments, the fetal cellular DNA may be obtained fromother fetal cell types circulating in the maternal blood.

In some embodiments, the method also involves obtaining mother-and-fetusmixed cfDNA from the pregnant mother. See block 606. The cfDNA includesat least one sequence that maps to the at least one sequence ofinterest. In some embodiments, the cfDNA is obtained from the plasma ofa blood sample from the mother. In some embodiments, the same bloodsample also provides the fetal NRBC as the source of the fetal cellularDNA. Of course, the cellular DNA and cfDNA may also be obtained fromdifferent samples of the same mother.

In some embodiments, the method applies an indicator of the source ofDNA as being from the fetal cellular DNA or from the cfDNA. In someembodiments, this indicator comprises a first library identifier and asecond library identifier. In some embodiments, the process involvespreparing a first sequencing library of fetal cellular DNA obtained fromoperation 602, wherein the first sequencing library is identifiable by afirst library identifier. Block 604. In some embodiments, the firstlibrary identifier is a first index sequence that is identifiable indownstream sequencing steps. In some embodiments, the indicator of thesource of DNA also comprises a second sequencing library of the cfDNAidentifiable by a second library identifier. Block 608. In preparingsequence libraries, the method may involve incorporating indexes to eachof said sequence libraries, wherein the indexes incorporated to saidfirst library differ from the indexes incorporated to said secondlibrary. The indexes contain unique sequences (e.g., bar codes) that areidentifiable in downstream sequencing steps, thereby providing anindicator of the source of the nucleic acids.

In some embodiments, the indicator of the source of DNA may be providedby other methods such as size separation.

In some embodiments, the method proceeds by combining at least a portionof the fetal cellular DNA of the first sequencing library and at least aportion of the cfDNA of the second sequencing library to provide amixture of the first and second sequencing libraries. See block 610.

In FIG. 6, preparation of the first sequencing library and the secondsequencing library is shown as two separate branches of the workflow,and the prepared libraries are combined to obtain a mixture of the firstand second sequencing libraries. However, in some embodiments the twolibraries are indexed separately at the beginning, then furtherprocessed in a combined sample. In some embodiments, the method involvesfurther processing the combined sample to prepare or modify sequencinglibraries. In some embodiments, the further processing involvesincorporating sequencing adaptors (e.g., paired end primers) formassively parallel sequencing.

In some embodiments, the method then proceeds with sequencing at least aportion of the mixture of the first and second sequencing libraries toprovide a first plurality of sequence tags identifiable by the firstlibrary identifier and a second plurality of sequence tags identifiableby second library identifier. See block 612. In some embodiments, thesequence reads are then mapped to a reference sequence containing thesequence of interest, thereby providing sequence tags mapped to thesequence of interest. In some embodiments, the sequence of interest mayidentify the presence of an allele. In some embodiments, the sample hasbeen selectively enriched for the sequence of interest.

In some embodiments, instead of or in addition to selective enrichmentof the sequence of interest before sequencing, the sample may beamplified by whole genome amplification. In some of these embodiments,the sequence reads are aligned to a reference genome comprising asequence of interest (e.g., chromosome, chromosome segment) that aretypically longer than in the embodiment with selective enrichmenttargeting shorter sequences of interest (e.g., SNPs, STRs, and sequencesof up to kb in size). The sequence reads mapping to the sequence ofinterest provide sequence tags for the sequence of interest, which canbe used to determine a genetic condition, e.g., aneuploidy, related tothe sequence of interest.

In some embodiments, the method applies massively parallel sequencing.Various sequencing techniques may be used, including but not limited to,sequencing by synthesis and sequencing by ligation. In some embodiments,sequencing by synthesis uses reversible dye terminators. In someembodiments, single molecule sequencing is used.

In some embodiments, the method further involves analyzing the first andsecond pluralities of sequence tags to determine the at least onesequence of interest. See block 614. At least a portion of the pluralityof sequence tags map to the at least one sequence of interest. In someembodiments, the method determines the presence or abundance of sequencetags mapping to the sequence of interest. This may include determiningCNV (e.g., aneuploidy) and non-NCV abnormality. Particularly, the methodmay determine the relative amounts of two alleles in each of the cfDNAand cellular DNA. In some embodiments, the method may detect that thefetus has a genetic disorder by determining that the fetus is homozygousof a disease causing allele of a disease related gene wherein the motheris heterozygous of the allele.

In some embodiments, the method starts with cellular DNA and cfDNA inseparate reaction environments, e.g., test tubes. In some embodiments,the method involves enriching wild-type and mutant regions using probesthat target both alleles of disease related gene(s) and have differentindices for cellular DNA and cfDNA, the indices are incorporated intothe targeted sequences in the separate reaction environment. The methodfurther involves mixing the cellular DNA and cfDNA with enrichedtargeted regions and amplifying the DNA using universal PCR primers. Insome embodiments, whole genome amplification instead of targetedsequence amplification is applied. The amplified product will besequencing-ready libraries of both cellular DNA of the fetus and cfDNAfor the mother and fetus. The sequencing results may then be used todetermine a sequence of interest for the fetus. In some embodiments,determining the sequence of interest provides information for detectinga CNV or non-CNV chromosomal anomaly involving the sequence of interest.In some embodiments, the method may determine the zygosity of the fetusand/or fetal fraction of the cfDNA.

In some embodiments, the method further involves determining a pluralityof training sequences from the cfDNA and the cellular DNA, which can beused to determine a CNV or non-CNV chromosomal anomaly involving asequence of interest. Some embodiments further use the sequenceinformation obtained from the cellular DNA to determine the fetalfraction of the cfDNA. The methods exemplified in FIG. 6 and set forthabove with respect to DNA can be carried out for other nucleic acids(e.g. mRNA) as well.

Obtain cfDNA and Fetal Cellular DNA

In various embodiments, mother-and-fetus mixed cfDNA and fetal cellularDNA are obtained from maternal peripheral blood to provide the geneticmaterials, as respectively shown in block 602 and block 606 of FIG. 6.The genetic materials are used to generate two identifiable libraries asrespectively shown in block 604 and block 608 of FIG. 6. The twolibraries are then combined for further downstream processing andanalyses. Various methods may be used to obtain cfDNA and fetal cellularDNA. Two processes are described below as examples to illustrateapplicable methods for obtaining cfDNA and fetal cellular DNA fordownstream processing and analyses.

A Process of Obtaining DNA Using Fixed Blood

Fetal cellular DNA and mixed cfDNA may be obtained from fixed or unfixedblood samples. Maternal peripheral blood samples can be collected usingany of a number of various different techniques. Techniques suitable forindividual sample types will be readily apparent to those of skill inthe art. For example, in certain embodiments, blood is collected inspecially designed blood collection tubes or other container. Such tubesmay include an anti-coagulant such as ethylenediamine tetracetic acid(EDTA) or acid citrate dextrose (ACD). In some cases, the tube includesa fixative. In some embodiments, blood is collected in a tube thatgently fixes cells and deactivates nucleases (e.g., Streck Cell-free DNABCT tubes). See US Patent Application Publication No. 2010/0209930,filed Feb. 11, 2010, and US Patent Application Publication No.2010/0184069, filed Jan. 19, 2010 each previously incorporated herein byreference.

FIG. 7 depicts a flowchart of a process 700 to obtain mother-and-fetuscfDNA and fetal cellular DNA using a fixed whole blood sample obtainedfrom a pregnant mother. Of course, the process may be modified to usetwo samples from the same pregnant mother, with one sample providingcfDNA and one providing cellular DNA. Process 700 begins with mixing amild fixative with a maternal blood sample that includes cellular DNAand cfDNA. Block 702. The cellular DNA may originate from maternal cellsand/or fetal cells. The blood sample can be collected by any one of manyavailable techniques. Such techniques should collect a sufficient volumeof sample to supply enough cfDNA to satisfy the requirements of thesequencing technology, and account for losses during the processingleading up to sequencing.

In certain embodiments, blood is collected in specially designed bloodcollection tubes or other container. Such tubes may include ananti-coagulant such as ethylenediamine tetracetic acid (EDTA) or acidcitrate dextrose (ACD). In some cases, the tube includes a fixative. Insome embodiments, blood is collected in a tube that gently fixes cellsand deactivates nucleases (e.g., Streck Cell-free DNA BCT tubes). See USPatent Application Publication No. 2010/0209930, filed Feb. 11, 2010,and US Patent Application Publication No. 2010/0184069, filed Jan. 19,2010 each previously incorporated herein by reference.

Generally, it is desirable to collect and process cfDNA that isuncontaminated with DNA from other sources such as white blood cells.Therefore, white blood cells can be removed from the sample and/ortreated in a manner that reduces the likelihood that they will releasetheir DNA.

Process 700 then proceed to separate a plasma fraction from anerythrocyte fraction of the fixed blood sample. In some embodiments, toseparate the plasma fraction from the erythrocyte fraction, the processcentrifuges the blood sample at a low speed, then aspirates andseparately saves the plasma, buffy coat, and erythrocyte fractions. Seeblock 704.

In some embodiments, the blood sample is centrifuged, sometimes formultiple times. The first centrifugation step applies a low speed toproduce three fractions: a plasma fraction on top, a buffy coatcontaining leukocytes, and an erythrocyte fraction on the bottom. Thisfirst centrifugation process is performed at relatively low g-force inorder to avoid disrupting the hematocytes (e.g. leukocytes, nucleatederythrocytes, and platelets) to a point where their nuclei break apartand release DNA into the plasma fraction. Density gradientcentrifugation is typically used. If this first centrifugation step isperformed at too high of an acceleration, some DNA from the leukocyteswould likely contaminate the plasma fraction. After this centrifugationstep is completed, the plasma fraction and erythrocyte fraction areseparated from each other and can be further processed.

The plasma fraction can be subjected to a second higher speedcentrifugation to size fractionate DNA, removing larger particulatesfrom the plasma, leaving cfDNA in the plasma. See block 706. In thisstep, additional particulate matter from the plasma is pelleted as asolid phase and removed. This additional solid material may include someadditional cells that also contain DNA that would otherwise contaminatethe cell free DNA that is to be analyzed. In some embodiments, the firstcentrifugation is performed at an acceleration of about 1600 g and thesecond centrifugation is performed at an acceleration of about 16,000 g.

While a single centrifugation process from normal blood is possible toobtain cfDNA, such process has been found to sometimes produce plasmacontaminated with white blood cells. Any DNA isolated from this plasmawill include some cellular DNA. Therefore, for cfDNA isolation fromnormal blood, the plasma may be subjected to a second centrifugation athigh-speed to pellet out any contaminating cells.

After removing larger sized particulates from the plasma by sizefractionation, the process 700 proceeds to isolate/purify cfDNA from theplasma. See block 708. In some embodiments, the isolation can beperformed by the following operations.

A. Denature and/or degrade proteins in plasma (e.g. contact withproteases) and add guanidine hydrochloride or other chaotropic reagentto the solution (to facilitate driving cfDNA out of solution)

B. Contact treated plasma with a support matrix such as beads in acolumn. cfDNA comes out of solution and binds to matrix.

C. Wash the support matrix.

D. Release cfDNA from matrix and recover the cfDNA for downstreamprocess (e.g., indexed library preparation) and statistical analyses.

After a plasma fraction is collected as described, the cfDNA isextracted. Extraction is actually a multistep process that involvesseparating DNA from the plasma in a column or other solid phase bindingmatrix. The extracted cfDNA usually includes both maternal and fetalcfDNA. Depending on the pregnancy stage and physiological condition ofthe mother and the fetus, the cfDNA can include up to 10% of fetal DNAin some examples.

The first part of this cfDNA isolation procedure involves denaturing ordegrading the nucleosome proteins and otherwise taking steps to free theDNA from the nucleosome. A typical reagent mixture used to accomplishthis isolation includes a detergent, protease, and a chaotropic agentsuch as guanine hydrochloride. The protease serves to degrade thenucleosome proteins, as well as background proteins in the plasma suchas albumin and immunoglobulins. The chaotropic agent disrupts thestructure of macromolecules by interfering with intramolecularinteractions mediated by non-covalent forces such as hydrogen bonds. Thechaotropic agent also renders components of the plasma such as proteinsnegative in charge. The negative charge makes the medium somewhatenergetically incompatible with the negatively charged DNA. The use of achaotropic agent to facilitate DNA purification is described in Boom etal., “Rapid and Simple Method for Purification of Nucleic Acids”, J.Clin. Microbiology, v. 28, No. 3, 1990.

After this protein degradation treatment, which frees, at leastpartially, the DNA coils from the nucleosome proteins, the resultingsolution is passed through a column or otherwise exposed to supportmatrix. The cfDNA in the treated plasma selectively adheres to thesupport matrix. The remaining constituents of the plasma pass throughthe binding matrix and are removed. The negative charge imparted tomedium components facilitates adsorption of DNA in the pores of asupport matrix.

After passing the treated plasma through the support matrix, the supportmatrix with bound cfDNA is washed to remove additional proteins andother unwanted components of the sample. After washing, the cfDNA isfreed from the matrix and recovered. Notably, this process loses asignificant fraction of the available DNA from the plasma. Generally,support matrixes have a high capacity for cfDNA, which limits the amountof cfDNA that can be easily separated from the matrix. As a consequence,the yield of cfDNA extraction step can be quite low. Typically, theefficiency is well below 50% (e.g., it has been found that the typicalyield of cfDNA is 4-12 ng/ml of plasma from the available ˜30 ng/mlplasma).

Other methods may be used to obtain cfDNA from a maternal blood samplewith higher yield. One example is further described here. For instance,in one embodiment, a device can be used to collect 2-4 drops ofpatient/donor blood (100-200 ul) and then separate the plasma from thehematocrit using a specialized membrane. The device can be used togenerate the required 50-100 μl of plasma for NGS library preparation.Once the plasma has been separated by the membrane, it can be absorbedinto a pretreated medical sponge. In certain embodiments, the sponge ispretreated with a combination of preservatives, proteases and salts to(a) inhibit nucleases and/or (b) stabilize the plasma DNA untildownstream processing. Products such as Vivid Plasma Separation Membrane(Pall Life Sciences, Ann Arbor, Mich.) and Medisponge 50PW (Filtronatechnologies, St. Charles, Mich.) can be used. The plasma DNA in themedical sponge can be accessed for NGS library generation in a varietyof ways. (a) Reconstitute and extract that plasma from the sponge andisolate DNA for downstream processing. Of course, this approach may havelimited DNA recovery efficiency. (b) Utilize the DNA-binding propertiesof the medical sponge polymer to isolate the DNA. (c) Conduct directPCR-based library preparation using the DNA that is bound to the sponge.This may be conducted using any of the cfDNA library preparationtechniques described herein.

The purified cfDNA obtained from operation 708 can be used to prepare alibrary for sequencing. To sequence a population of double-stranded DNAfragments using massively parallel sequencing systems, the DNA fragmentsmust be flanked by known adapter sequences. A collection of such DNAfragments with adapters at either end is called a sequencing library.Two examples of suitable methods for generating sequencing librariesfrom purified DNA are (1) ligation-based attachment of known adapters toeither end of fragmented DNA, and (2) transposase-mediated insertion ofadapter sequences. There are many suitable massively parallel sequencingtechniques. Some of these are described below.

Note that operations 702-708 described so far for process 700 depictedin FIG. 7 largely overlap with operations 802-808 in process 800 of FIG.8 described below.

Process 700 also provides fetal cellular DNA from the maternal bloodsample, which makes use of the erythrocyte fraction obtained from thelow-speed centrifugation of operation 704. In some embodiments, theprocess involves lysing the erythrocytes in the erythrocyte fractionDNA, the product from which includes both cfDNA and cellular DNA. Seeblock 710. Next, process 700 proceeds by centrifuging the sample to sizefractionate DNA, allowing the separation of cfDNA and cellular DNA,since cfDNA is much smaller in size than cellular DNA as describedabove. See block 712. In some embodiments, this centrifugation operationmay be similar to the centrifugation of operation 706, performed at16,000 g. In some implementations, the cfDNA obtained from theerythrocyte fraction may optionally be combined with the cfDNA obtainedfrom the plasma fraction for downstream processing. See block 708.

Process 700 allows obtaining cellular DNA from the erythrocyte fraction.See block 714. The cellular DNA obtained from the erythrocytes fractionlargely originates from NRBCs. During pregnancy, most of the NRBC thatare present in the maternal blood stream are those that have beenproduced by the mother herself. See Wachtel, et al., Prenat. Diagn.18:455-463 (1998). In some instances, the cellular DNA include up to 50%of fetal cellular DNA. For example, the cellular DNA may include 70% ofmaternal DNA and 30% of fetal DNA as shown by Wachtel et al.

In some embodiments, process 700 proceeds by isolating the fetalcellular DNA from maternal cellular DNA. See block 706. Various methodsmay be applied to separate the two sources of cellular DNA by takingadvantage of the different characteristics of the two sources of DNA.See block 716. For instance, it has been shown that fetal DNA tends tohave a higher state of methylation than maternal DNA. Therefore,mechanisms that differentiate methylation may be used to separate fetalcellular DNA from maternal cellular DNA. See, e.g., Kim et al., Am JReprod Immunol. 2012 July; 68(1):8-27, for different methylationcharacteristics of maternal versus fetal cells.

Additionally, FISH can be used to detect and localize specific DNA orRNA targets from fetal cells. Some embodiments may ascertain fetalorigin by FISH that identifies fetal specific DNA markers. Therefore,process 700 allows one to obtain fetal cellular DNA, which can then befurther processed and analyzed. See block 718.

A Process of Obtaining DNA Using Unfixed Blood

The disclosure also provides methods for obtaining fetal cellular DNAand mixed cfDNA using unfixed blood samples. FIG. 8 is a flowchartshowing a process of such a method. The operations for obtaining cfDNAdepicted in FIG. 8 largely overlap with those in the process depicted inFIG. 7. Therefore blocks 704, 706 and 708 mirror blocks 804, 806 and808.

Briefly, process 800 starts by mixing an anti-coagulant such as EDTA orACD with the maternal blood sample without using a fixative. See block802. Process 800 proceeds by separating a plasma fraction and anerythrocyte fraction from the blood sample by centrifugation. See block804. As in block 804, the centrifugation may be performed at alower-speed, such as 1600 g. The sample is then aspirated, and plasma,buffy coat, and the erythrocyte fractions are separately saved. Theplasma fraction obtained from operation 804 and then undergo a secondcentrifugation at a higher speed such as 16,000 g to size fractionateDNA, spinning out larger particulates and leaving smaller cfDNA in theplasma. See block 806. Process 800 provides means to obtain cfDNA fromthe plasma that can be used for further processing and analysis. Seeblock 808.

Operations 810-818 of process 800 allow isolation of fetal NRBCs formthe erythrocyte fraction, and obtaining fetal cellular DNA from theisolated fetal NRBCs. Operation 810 involves adding isotonic buffer tothe erythrocyte fraction. Then the process proceeds by centrifugation topellet intact erythrocytes. See block 814. In some embodiments, thiscentrifugation is performed at a lower speed than that in operation 806in order to avoid rupturing the erythrocytes. The supernatant from thiscentrifugation includes cfDNA that can be combined with the cfDNAobtained from the plasma fraction for downstream processing andanalysis. See block 808. The pellet, or compacted precipitant, includesintact erythrocytes from both the mother and the fetus, wherein theerythrocytes from the mother include a large portion of enucleated RBCsand a small number of NRBCs.

In some embodiments, process 800 proceeds by washing erythrocyte pelletwith isotonic buffer, then centrifuging to collect maternal enucleatedRBCs and NRBCs. The NRBCs include both maternal and fetal NRBCs, with upto 30% of fetal cells in some embodiments as discussed above. Process800 then proceeds by isolating fetal NRBCs from maternal cells. Seeblock 818. One can then obtain fetal cellular DNA from the isolatedfetal NRBCs. See block 820.

Isolate Fetal NRBC and Fetal Cellular DNA

In various embodiments, such as operations 818 and 820 of process 800depicted in FIG. 8, fetal NRBCs are isolated from maternal cells, andfetal cellular DNA is obtained from the isolated fetal NRBCs. Variouscombinations of methods may be applied to isolate NRBCs from maternalcells. In some embodiments, the methods can include various combinationsof cell sorting with magnetic particles or flow cytometry, densitygradient centrifugation, size-based separation, selective cell lysis, ordepletion of unwanted cell populations. Often, these methods alone arenot effective because each method may be able to remove some unwantedcells but not all. Therefore combination of methods can be used toisolate the desired fetal NRBCs.

In some embodiments, isolation of fetal NRBCs is combined withenrichment of the fetal NRBCs by one or more methods known in the art ordescribed herein. The enrichment increases the concentration of rarecells or ratio of rare cells to non-rare cells in the sample. In someembodiments, when enriching fetal cells from a maternal peripheralvenous blood sample, the initial concentration of the fetal cells may beabout 1:50,000,000 and it may be increased to at least 1:5,000 or 1:500.Enrichment can be achieved by one or more types of separation modulesdescribed herein or in the prior art. See, e.g., U.S. Pat. No. 8,137,912for some techniques for enrichment of fetal cells, which is incorporatedby reference in its entirety. Multiple separation modules may be coupledin series for enhanced performance.

In some embodiments, the fetal cellular DNA used for downstreamprocessing is obtained from one or more fetal NRBCs in the blood of thepregnant mother. In some embodiments, the method separates the fetalNRBCs from maternal erythrocytes in a cellular component of a bloodsample of the pregnant mother. In some embodiments, separating the fetalNRBCs from the maternal erythrocytes comprises differentially lysingmaternal erythrocytes. In some embodiments, separating the fetal NRBCsfrom the maternal erythrocytes comprises size-based separation and/orcapture-based separation. The capture-based separation may comprisecapturing the fetal NRBCs through binding one or more cellular markersexpressed by fetal NRBCs. Preferably, the one or more cellular markerscomprise a surface marker expressed by fetal NRBCs but not, or to alesser degree, by maternal NRBCs. In some embodiments, the capture-basedseparation comprise binding magnetically responsive particles to fetalNRBCs, wherein the magnetically responsive particles have an affinity toone or more cellular markers expressed by fetal NRBCs. In someembodiments, the capture-based separation is performed by an automatedimmunomagnetic separation device, for example, as described in U.S. Pat.No. 8,071,395, which is incorporated herein by reference. In someembodiments, the capture-based separation comprises binding fluorescentlabels to fetal NRBCs, wherein the fluorescent labels have an affinityto one or more cellular markers expressed by fetal NRBCs.

In various embodiments, cell surface markers expressed on fetal NRBCsare used for affinity based separation. For instance, some embodimentsmay use anti-CD71 to attach magnetic or fluorescent probes totransferrin receptors, which probes provide a mechanism formagnetic-activated cell sorting (MACS) or fluorescence-activated cellsorting (FACS). Cells from very early developmental stages can beisolated from umbilical cord blood using CD34. To enrich and identifyerythroid cells from later developmental stages, surface markers such asCD71, glycophorin A, CD36, antigen-i, and intracellularly expressedhemoglobins may be used. Soybean agglutinin (SBA) may be used to isolatefetal NRBCs from the blood of pregnant mothers.

Many of the above surface markers are not exclusive to fetal NRBCs.Instead, they are also expressed to various degrees on maternal cells.Recently, monoclonal anti-bodies have been identified with affinity tofetal NRBCs but not to maternal bloods. For instance, Zimmermann et al.identified monoclonal antibody clones 4B8 and 4B9 that has specificaffinity to fetal NRBCs. Experimental Cell Research, 319 (2013),2700-2707. The mAb 4B8, 4B9 and other similar mABs may be used toprovide binding mechanism for MACS or FACS to isolate fetal NRBCs.Magnetism based cell separation may be implemented as a MagSweeperdevice, which is an automated immunomagnetic separation technology asdisclosed in U.S. Pat. No. 8,071,395, which is incorporated by referencein its entirety. In some implementations, the MagSweeper can enrichcirculating rare cells, e.g., fetal NRBCs in maternal blood, by an orderof 10⁸-fold increase in concentration.

The fetal origin of isolated cells can be indicated by PCR amplificationof Y chromosome specific sequences, by fluorescence in situhybridization (FISH), by detecting ϵ-globin and γ-globin, or bycomparing DNA-polymorphisms with STR-markers from mother and child. Someembodiments may use these indicators to separate fetal NRBCs from othercells, e.g., implemented as imaging-based separation mechanism byvisualizing the indicator or as affinity-based separation mechanism byhybridizing with the indicator.

FIG. 9 is a flowchart showing process 900 for isolating fetal NRBCs froma maternal blood sample according to some embodiments of the disclosure.Process 900 relates to process 800 in that process 900 provides oneexample of how operation 818 in FIG. 8 may be accomplished. Process 900starts by obtaining RBCs from maternal blood sample, see block 902, suchas using one or more density gradient centrifugations as described inthe steps leading to step 816.

The process then proceeds to remove maternal enucleated RBCs and NRBCsfrom the RBCs by selectively lysing maternal erythrocytes usingacetazolamide and lysing solutions containing NH₄ ⁺ and HCO₃ ⁻. Seeblock 904. Erythrocytes can be quickly disrupted in lysing solutionscontaining NH₄ ⁺ and HCO₃ ⁺. Carbonic anhydrase catalyzes this hemolysisreaction, and is at least 5-fold lower in fetal cells than adult cells.Therefore the hemolytic rate is slower for fetal cells. Thisdifferential of hemolysis is augmented by acetazolamide, which is aninhibitor of carbonic anhydrase, and which penetrates fetal cell about10 times faster than adult cells. Therefore the combination ofacetazolamide and lysing solutions containing NH₄ ⁺ and HCO₃ ⁺selectively lyses the maternal cells while sparing the fetal cells.

In one embodiment, the differential lyses may be performed as in thefollowing example. The RBCs are centrifuged (e.g., 300 g, 10 min),re-suspended in phosphate-buffered saline (PBS) with acetazolamide, andincubated at room temperature for 5 min. Two and one half milliliters oflysis buffer (10 mM NaHCO₃, 155 mM NH₄Cl) is added and the cells areincubated for 5 min, centrifuged, re-suspended in lysis buffer,incubated for 3 min, and centrifuged.

After the selectively lysing maternal RBCs, lysed cells may be removedby centrifugation. In some embodiments, the process proceeds to labelfetal NRBCs with magnetic beads coated with an antibody that binds to acell surface marker expressed on the fetal NRBCs. See block 906. One ormore of the surface markers expressed on fetal NRBCs described above maybe the target for binding. In some embodiments, mAb 4B8, mAb 4B9, oranti-CD71 may be used as the antibody that binds to the surface of fetalNRBCs. The magnetic beads provides a means for magnetic separationmechanism to capture the fetal NRBCs, which are then selectivelyenriched. In some embodiments, the process proceeds to label the fetalNRBCs with a fluorescent label, e.g., oligonucleotides (“oligos”) boundto fluorescein or rhodamine, which oligos bind to mRNA of markers offetal NRBCs. In some embodiments, the fluorescent label binds to themRNA of fetal hemoglobin, e.g., ϵ-globin and γ-globin.

Process 900 proceeds to enrich the fetal NRBCs using magnetic separationdevice such as the MagSweeper described above, which captures the NRBCsthrough the magnetic beads selectively attached to the NRBCs. See block910. Finally, process 900 achieves isolation of fetal NRBCs using animage guided cell isolation device such as a FACS sensitive to thefluorescent label attached to the fetal NRBCs in operation 908. Seeblock 912. The isolated fetal NRBCs may then be used to prepare anindexed fetal cellular DNA library. Some embodiments of the preparationof the indexed library are further described below.

In many embodiments, fetal NRBCs are first isolated from maternal RBCsand other cell types. Then fetal cellular DNA is obtained from theisolated fetal NRBCs. However, in some embodiments, fetal cellular DNAmay be obtained by selectively lysing fetal NRBCs (as opposed to lysingthe maternal cells). For example, fetal cells can be selectively lysedreleasing their nuclei when a blood sample including fetal cells iscombined with deionized water. Such selective lysis of the fetal cellsallows for the subsequent enrichment of fetal DNA using, e.g., size oraffinity based separation.

Sequence Library Preparation Involving Fetus-Only Cellular DNA

The preparation of sequencing Libraries involving fetus-only cellularDNA is similar to that involving mother-only cellular DNA describedabove. However, some aspects are specific to preparing fetus-onlycellular DNA, which are further described below.

In some embodiments, the method involves enriching wild-type and mutantregions using probes that target both alleles of disease related gene(s)and have different indices for cellular DNA and cfDNA, the indices areincorporated into the targeted sequences in the separate reactionenvironment. The method further involves mixing the cellular DNA andcfDNA with enriched targeted regions and amplifying the DNA usinguniversal PCR primers. The amplified product will be sequencing-readytargeted libraries of both cellular DNA for the mother and cfDNA for themother and fetus. In some embodiments, whole genome amplification isperformed without selective enrichment of target sequences. Thesequencing results may then be used to determine a sequence of interestfor the fetus. The method may determine CNV or zygosity of the fetus. Insome embodiments, the method determines the fetal fraction of the cfDNA.

In some embodiments, fetal NRBCs are first isolated using variousmethods. Then the embodiments use the isolated fetal NRBCs to prepare anindexed library. FIG. 10 is a flowchart showing a process 1000 forpreparing an indexed library of fetal cellular DNA. In some embodiments,process 1000 starts by providing one or more isolated fetal NRBCs. Seeblock 1002. The fetal NRBCs may be isolated and obtained using methodssuch as the one described in FIG. 9. Then process 1000 proceeds bylysing the fetal NRBCs to release the fetal cellular DNA. See block1004. In some embodiments, process 1000 optionally includes amplifyingthe fetal cellular DNA using methods described herein or known in theart. See block 1006. In some embodiments, process 1000 applies Tn5transposase-mediated transposon tagging to create a second indexedsequencing library from the fetal cellular DNA. See block 1008. In someembodiments, the index sequence for the sequencing library may beincorporated by alternative methods as described elsewhere herein orknown in the art. An example using a site selective extension primer toincorporate the index sequence is described below.

In some embodiments, the method for determining a sequence of interestfor the fetus involves preparing an indexed library using the cfDNAobtained from a maternal blood sample. The sequencing library of thecfDNA has a different index than the library of the fetal cellular DNA.FIG. 11 is a flowchart showing a process 1100 for preparing an indexedlibrary of cfDNA, including steps for obtaining the cfDNA from amaternal blood sample, which blood sample may also provide fetalcellular DNA in some embodiments. Process 1100 starts by centrifuging amaternal blood sample with a low-speed spin for 15 min. at 4° C. Thecentrifugation may be performed and 16,000 g. See block 1102. Thecentrifugation separates the blood sample into a plasma fraction, abuffy coat fraction, and an erythrocyte fraction. The process proceedsby obtaining the plasma or serum from the sample. See block 1104. Theprocess further involves removing the upper layer of plasma, andspinning the plasma twice for 10 min. at 16,000 g to remove cellularsources of DNA, which are larger in size than cfDNA and are pelleted bythe high-speed centrifugation. See block 606. The process then proceedsby obtaining mother-and-fetus cfDNA from the plasma or serum fractionusing methods described herein or known in the art. See block 1108. Insome embodiments, process 1100 applies Tn5 mediated transposon taggingto create an indexed sequencing library from the cfDNA. See block 1110.In the preferred embodiments, the sequencing library of the cfDNA has adifferent index sequence from that of the fetal cellular DNA library. Insome embodiments, the index sequence for the sequencing library may beincorporated by alternative methods such as the method shown in FIG. 12.

Example Workflow for Preparing Sequence Libraries

Various methods may be used to prepare libraries indexed separately formother-and-fetus cfDNA and fetal cellular DNA. Plasma cfDNA from apregnant woman is a mixture comprising mostly of mother DNA with acertain fraction of fetal DNA. An accurate and precise determination ofthe % fetal DNA in maternal plasma cfDNA is desirable in non-invasiveprenatal testing, especially for samples with low fetal fractions. Onecommonly used method for determining fetal fraction interrogates aplurality of high heterozygocity SNPs and studies allelic frequencydifferences between mother and fetus. Data analysis can be challengingwhen one studies SNPs only in cfDNA, since “true” genotype of the motheralone or the fetus alone is not known. Some embodiments disclosed hereinprovide a means to determine the fetal zygosity genotype of the fetus,at least for some SNPs that are detectable from the relatively rarefetal NRBCs.

FIG. 12 shows a diagram of a workflow according to some embodiments ofthe invention, wherein the incorporation of index sequences occur withtwo primers introducing two adaptor segments. This workflow presents analternative to the workflow of using Tn5 transposase-mediated transposontagging to incorporate indexes, such as those depicted in FIGS. 10 and11. FIG. 12 presents a workflow where the cellular and cell-free DNAsare indexed independently during an early PCR step that alsointerrogates a plurality of SNP-sites in a multiplexed manner. Theindexing at this stage is called “source index” since it differentiatesbetween fetal cellular DNA and the mixed cfDNA within the same patientsample.

As shown in FIG. 12, the source index 1 sequence is incorporated intothe fetal cellular DNA, and the source index 2 is incorporated into thecfDNA, when the cellular DNA and the cfDNA are processed separately. Inthis indexing stage, two primers are applied, both targeting the SNP ofinterest diagram, which is shown as a dot at the center of the twosequences. The primers to the left of the SNP include a sequence indexas well as about one half of a paired end adaptor (e.g., the P5 adaptorfor the Illumina sequencing platform). Only about half of a sequencingadaptor is introduced by the primer to avoid a long overhang, whichcould dehybridize too easily. The remaining portion of the adaptor isintroduced at later processing stages. The primers to the right of theSNP in the depicted example include about one half of second paired endadaptor (e.g., a P7 adaptor for the Illumina sequencing platform). TheP5 and P7 adaptors allow library fragments to anneal to theircomplementary oligos on the flowcell surface of the Illumina sequencer,which adaptors are necessary to perform bridge amplification andclustering.

One option to consider at the indexing stage is to keep the number ofamplification cycles minimal, so as to minimize or avoid introduction ofbias between the two samples. In the depicted workflow, amplicons areindependently cleaned using Solid Phase Reversible Immobilisation(SPRI), then eluted in equal volume and pooled. The amplicons are thenmixed together and then subjected to universal PCR at the same time in asingle tube. The universal PCR introduces a second index at the P7 end,which second index can be used to multiplex patient samples duringsequencing. During the universal PCR, P5 and P7 adaptors are extended tofull length. By the end of PCR, library fragments are prepared toinclude all sequences necessary for multiplexed sequencing. Libraryfragments are then SPRI-cleaned and ready for sequencing.

Since each kind of DNA from the same patient sample is source-indexeduniquely, the downstream data from a given patient can be identified asarising from fetal cellular DNA or from the mixed mother-fetus cfDNA.

In conventional methods using only cfDNA, only those SNPs for which themother is homozygous and the fetus is heterozygous constitute“informative SNPs.” SNPs where the mother may be heterozygous and thefetus is homozygous are not easily used without understanding theinherent “noise” in the heterozygous calls for the mother. By genotypingfetal cellular DNA, the zygosity of a fetus may be obtained using datafrom the fetal cellular DNA. This zygosity can help to determine fetalfraction (FF) of cfDNA using both “informative” and “uninformative”data. The fetal fraction determined this way has higher confidenceinterval than methods using cfDNA alone. Furthermore, this more accuratemeasure of FF allows for better estimate of CNV.

Samples

Samples that are used for determining one or more sequences of interestcan include samples taken from any cell, tissue, or organ. In someembodiments, samples are used to determine the presence, abundance, copynumber or copy number variation of a sequence of interest. In someembodiments, the samples contain nucleic acids that are that are presentin cells and/or nucleic acids that are “cell-free” (e.g., cfDNA). Insome embodiments, the nucleic acids contain cellular DNA of the motheronly and cfDNA of both the mother and the fetus carried by the mother.In some embodiments, the mother-only cellular DNA is obtained from abuffy coat component of a blood sample of the mother, the cellular DNAbeing from maternal white blood cells or leukocytes. In someembodiments, the mother-and-fetus mixed cfDNA is obtained from a plasmacomponent of a blood sample of the mother.

In some embodiments, the mother-only cellular DNA and the mixed cfDNAare first processed separately to incorporate different index sequences,thereby providing an indicator of the source of the nucleic acids. Theindexed nucleic acids are then combined for further processing. In someembodiments, similar quantities of cellular DNA and cfDNA are combined.

Cell-free nucleic acids, including cell-free DNA, can be obtained byvarious methods known in the art from biological samples including butnot limited to plasma, serum, and urine (see, e.g., Fan et al., ProcNatl Acad Sci 105:16266-16271 [2008]; Koide et al., Prenatal Diagnosis25:604-607 [2005]; Chen et al., Nature Med. 2: 1033-1035 [1996]; Lo etal., Lancet 350: 485-487 [1997]; Botezatu et al., Clin Chem. 46:1078-1084, 2000; and Su et al., J Mol. Diagn. 6: 101-107 [2004]). Toseparate cell-free DNA from cells in a sample, various methodsincluding, but not limited to fractionation, centrifugation (e.g.,density gradient centrifugation), DNA-specific precipitation, orhigh-throughput cell sorting and/or other separation methods can beused. Commercially available kits for manual and automated separation ofcfDNA are available (Roche Diagnostics, Indianapolis, Ind.; Qiagen,Valencia, Calif.; or Macherey-Nagel, Duren, Del.). Biological samplescomprising cfDNA have been used in assays to determine the presence orabsence of chromosomal abnormalities, e.g., trisomy 21, by sequencingassays that can detect chromosomal aneuploidies and/or variouspolymorphisms.

In various embodiments the cfDNA present in the sample can be enrichedspecifically or non-specifically prior to use (e.g., prior to preparinga sequencing library). Specific enrichment of sample DNA refers toamplification of specific sequences contained in the sample, e.g.polymorphic sequences or sites. Non-specific enrichment of sample DNArefers to the whole genome amplification of the genomic DNA fragments ofthe sample that can be used to increase the level of the sample DNAprior to preparing a cfDNA sequencing library. Non-specific enrichmentcan be the selective enrichment of one of the two genomes present in asample that contain more than one genome. For example, non-specificenrichment can be selective of the fetal genome in a maternal sample,which can be obtained by known methods to increase the relativeproportion of fetal to maternal DNA in a sample. Alternatively,non-specific enrichment can be the non-selective amplification of bothgenomes present in the sample. For example, non-specific amplificationcan be of fetal and maternal DNA in a sample comprising a mixture of DNAfrom the fetal and maternal genomes. Methods for whole genomeamplification are known in the art. Degenerate oligonucleotide-primedPCR (DOP), primer extension PCR technique (PEP) and multipledisplacement amplification (MDA) are examples of whole genomeamplification methods. In some embodiments, the sample comprising themixture of cfDNA from different genomes is un-enriched for cfDNA of thegenomes present in the mixture. In other embodiments, the samplecomprising the mixture of cfDNA from different genomes isnon-specifically enriched for any one of the genomes present in thesample.

The sample comprising the nucleic acid(s) to which the methods describedherein are applied typically comprises a biological sample (“testsample”), e.g., as described above. In some embodiments, the nucleicacid(s) to be screened for one or more sequence of interest is purifiedor isolated by any of a number of well-known methods.

Accordingly, in certain embodiments the sample contains a purified orisolated polynucleotide, or it can contain samples such as a tissuesample, a biological fluid sample, a cell sample, and the like. Suitablebiological fluid samples include, but are not limited to blood, plasma,serum, sweat, tears, sputum, urine, sputum, ear flow, lymph, saliva,cerebrospinal fluid, ravages, bone marrow suspension, vaginal flow,trans-cervical lavage, brain fluid, ascites, milk, secretions of therespiratory, intestinal and genitourinary tracts, amniotic fluid, milk,and leukophoresis samples. In some embodiments, the sample is a samplethat is easily obtainable by non-invasive procedures, e.g., blood,plasma, serum, sweat, tears, sputum, urine, sputum, ear flow, saliva orfeces. In certain embodiments the sample is a peripheral blood sample,or the plasma and/or serum fractions of a peripheral blood sample. Inother embodiments, the biological sample is a swab or smear, a biopsyspecimen, or a cell culture. In another embodiment, the sample is amixture of two or more biological samples, e.g., a biological sample cancomprise two or more of a biological fluid sample, a tissue sample, anda cell culture sample. As used herein, the terms “blood,” “plasma” and“serum” expressly encompass fractions or processed portions thereof.Similarly, where a sample is taken from a biopsy, swab, smear, etc., the“sample” expressly encompasses a processed fraction or portion derivedfrom the biopsy, swab, smear, etc.

In one illustrative, but non-limiting embodiment, the sample is amaternal sample that is obtained from a pregnant female, for example apregnant woman. In this instance, the sample can be analyzed using themethods described herein to provide a prenatal diagnosis of potentialgenetic diseases or chromosomal abnormalities in the fetus. The maternalsample can be a tissue sample, a biological fluid sample, or a cellsample.

In another illustrative, but non-limiting embodiment, the maternalsample is a mixture of two or more biological samples, e.g., thebiological sample can comprise two or more of a biological fluid sample,a tissue sample, and a cell culture sample. In some embodiments, thesample is a sample that is easily obtainable by non-invasive procedures,e.g., blood, plasma, serum, sweat, tears, sputum, urine, milk, sputum,ear flow, saliva and feces. In some embodiments, the biological sampleis a peripheral blood sample, and/or the plasma and serum fractionsthereof. In other embodiments, the biological sample is a swab or smear,a biopsy specimen, or a sample of a cell culture. As disclosed above,the terms “blood,” “plasma” and “serum” expressly encompass fractions orprocessed portions thereof. Similarly, where a sample is taken from abiopsy, swab, smear, etc., the “sample” expressly encompasses aprocessed fraction or portion derived from the biopsy, swab, smear, etc.

In certain embodiments samples can also be obtained from in vitrocultured tissues, cells, or other polynucleotide-containing sources. Thecultured samples can be taken from sources including, but not limitedto, cultures (e.g., tissue or cells) maintained in different media andconditions (e.g., pH, pressure, or temperature), cultures (e.g., tissueor cells) maintained for different periods of length, cultures (e.g.,tissue or cells) treated with different factors or reagents (e.g., adrug candidate, or a modulator), or cultures of different types oftissue and/or cells.

Methods of isolating nucleic acids from biological sources are wellknown and will differ depending upon the nature of the source. One ofskill in the art can readily isolate nucleic acid(s) from a source asneeded for the method described herein. In some instances, it can beadvantageous to fragment the nucleic acid molecules in the nucleic acidsample. Fragmentation can be random, as achieved, for example, usingmechanical shearing, or it can be sequence-specific, as achieved, forexample, using restriction endonuclease digestion. Methods for randomfragmentation are well known in the art, and include, for example,limited DNAse digestion, alkali treatment and physical shearing. In oneembodiment, sample nucleic acids are obtained from cfDNA, which is notsubjected to fragmentation in vitro.

In some illustrative embodiments, the sample nucleic acid(s) areobtained as genomic DNA, which is subjected to fragmentation intofragments of approximately 150 or more, approximately 300 or more,approximately 400 or more, or approximately 500 or more base pairs, andto which NGS methods can be readily applied.

Determine Fetal Zygosity and Fetal Fraction

In some embodiments, the sequence information obtained from mother-onlycellular DNA and mother-fetus mixed cfDNA are used to determine fetalzygosity or fetal fraction. In some embodiments, the method may detectthat the fetus has a genetic disorder by determining that the fetus ishomozygous of a disease causing allele of a disease related gene whereinthe mother is heterozygous of the allele.

In certain implementations described herein, methods are provided fordetermining fetal fraction of maternal DNA using multiple DNA sequencereadings at sequence sites known to harbor one or more polymorphisms.Typically, though not necessarily, such polymorphisms are singlenucleotide polymorphisms (SNP). Other types of suitable polymorphismsinclude deletions, STRs (Short Tandem Repeats), insertions, indels(including microindels), etc. In certain embodiments, the polymorphismsites are found on a “reference sequence” as described below. In someembodiments, the polymorphism sites are discovered while aligningsequence tags to one another and/or a reference sequence.

Certain disclosed methods make use of the fact that a fetus's DNAsequences at the polymorphism sites under consideration may notcorrespond to those of its mother. For example, the mother's DNA at thesite of a particular SNP may be homozygous, while the fetus's version ofthe SNP will be heterozygous. Hence, a collection of sequence samplestaken for the SNP in question will be heterogeneous with the majority ofthe sequences containing the major allele and the remaining fractioncontaining the minor allele. The relative amounts of the major and minoralleles are determined by the fraction of fetal DNA in the sample.

It should be mentioned that in a homozygous sample both copies of agiven SNP or other polymorphism contain the same allele, while aheterozygous SNP or other polymorphism contains one copy of the majorallele and one copy of the minor allele. One knows, therefore, that DNAtaken exclusively from a heterozygous individual should contain 50% ofthe major allele and 50% of the minor allele. This knowledge can be usedin elucidating the fraction of fetal DNA.

In some implementations, the DNA taken from the mother's blood is readmany times, with the total number of reads mapping to a particular siteof a polymorphism being deemed the “coverage” of the polymorphism, andthe number of reads mapping to the minor allele for that polymorphismbeing deemed the minor allele count. The ratio of minor allele count tocoverage is useful for determining fetal fraction in variousimplementations.

Table 1 shows hypothetical data that can be obtained using the processesintroduced herein. The data assumes the following: (1) 10% fetalfraction, (2) measured maternal genomic DNA yields a 48% reading of ballele in heterozygous mothers, (3) maternal and fetal cfDNA have thesame % as the corresponding maternal genomic DNAs, and (4) maternalgenomic DNA yields 100% or 0% reading of b allele in homozygous mother(b and a respectively). Table 1 below shows the fraction of allele b forthe hypothetical data that can be obtained from training sequences.While one might expect that the cellular DNA sample would give a 50%reading for allele b in the heterozygous case, this is not often thecase. Biases introduced in the amplification and sequence techniquesfrequently cause the reading to vary from 50%. Such biases may beaddressed by using a training set of polymorphisms and data fromcellular DNA.

TABLE 1 Fraction of allele b for training sequences Allele b fractionZygosity case Training Mat. genomic mixed cfDNA mixed cfDNA Mother SNPDNA (measured) (actual) fetus 1. 48%  48%  50% Heterozygous Heterozygous2. 48% 48(0.9) + 100(0.1) = 50(0.9) + 100(.1) = Heterozygous 53.2% 55%Homozygous b 3. 100%  100(0.9) + 48(0.1) = 100(0.9) + 50(0.1) =Homozygous b 94.8% 95% Heterozygous 4. 100%  100% 100% Homozygous bHomozygous b 5.  0% 0(0.9) + (48)(0.1) = 0(0.9) + (50)(0.1) = Homozygousa 4.8% 5% Heterozygous 6.  0%  0%  0% Homozygous a Homozygous a

In some embodiments, the method de-convolves the sequencing data basedon indices, trained data analysis on the targeted control sites anddeduce fetal zygosity for a disease causing alleles. In someembodiments, the zygosity case is determined by comparing the relativeamounts of each of two alleles in cellular DNA and in cfDNA for thesequence of interest and training sequences. In some implementations,the training sequences may be any sequences with ascertainable zygosity,such as sequences in the sex chromosomes or other sequences havingpolymorphisms whose zygosity cases correlate with observable phenotypes.In some embodiments, the zygosity case for the fetus is determined bycomparing the relative amounts of DNA mapping to two or more alleles. Ingeneral, neither the fetal fraction of the cfDNA nor the zygosity casesof the sequence of interest and the training set sequences are known atthe beginning of the method. This information may be determined bymodeling the training set zygosities and fetal fraction and determiningwhich set of zygosities and fetal fraction best fit the data. Varioustechniques may be employed for this purpose. For example, a mixturemodel may be employed to determine a mean and optionally the variancefor each of the four zygosity cases encountered in the training set. Inspecific embodiments, this is the mean and variance associated with thefrequency of the minor allele in relation to the total number of countsfor a polymorphism under consideration (coverage). The mean values forcertain zygosity cases are directly related to the fetal fraction in thecfDNA. The application of mixture models to a training set ofpolymorphisms and thereby determine fetal fraction is described in U.S.patent application Ser. No. 13/445,778, [ARTEP002US] filed Apr. 12,2012, which is incorporated herein by reference in its entirety.

FIG. 13 shows a process for determining fetal zygosity of a sequence ofinterest using cellular DNA and cfDNA. This process may be used todetermine the fetus zygosity when the mother is heterozygous, which isdifficult with conventional method because the major allele and minorallele have similar counts in the cfDNA. The method uses cellular DNAand cfDNA sequence data obtained from two sequencing libraries processedtogether, which equalizes processing bias between two sequencinglibraries for the cellular DNA and cfDNA. The two libraries includedifferent indexes to identify the source of the genetic materials. Thisallows for comparison of data from the two libraries, which helps toimprove discrimination power between the fetus zygosity when the motheris heterozygous.

In some embodiments, the sequencing libraries are processed startingwith substantially the same or similar size and concentration cellularDNA and cfDNA in separate reaction environments, e.g., test tubes. Thisallows comparison of sequence tags from cellular DNA and cfDNA. In someembodiments this allows the subtraction of sequence tags obtained fromthe two libraries. In some embodiments, wild-type and mutant regions ofdisease related gene(s) are enriched using probes that target bothalleles of disease related gene(s) and have different indices forcellular DNA and cfDNA, the indices are incorporated into the targetedsequences in the separate reaction environment (e.g., separate tubes).Then the cellular DNA and cfDNA with enriched targeted regions are mixedtogether and amplified using universal PCR primers. The amplifiedproduct will be sequencing-ready targeted libraries of both cellular DNAfor the mother and cfDNA for the mother and fetus. The sequencingresults may then be used to determine the zygosity of the fetus and/orfetal fraction of the cfDNA.

In some embodiments, process 1300 for determining fetal zygosityinvolves obtaining a test-difference-value based on the difference ofcounts of test sequence tags derived from the mother-only cellular DNAvs. the cfDNA for each of or at least one of the two alleles, where thetest sequence tags map to the sequence of interest. See block 1302. Invarious embodiments, the test-difference-value may be obtained bysubtraction, division, or other operations that reflect the differenceof sequence tag counts. In some embodiments, the test-difference-valuemay be normalized.

In some embodiments, process 1300 also involves obtaining a plurality oftraining-difference-values based on the difference of counts of trainingsequence tags derived from the cellular DNA vs. the cfDNA, wherein thetraining sequence tags map to a plurality of training sequences. In someimplementations, the training sequences may be any sequences withascertainable zygosity, such as sequences in the sex chromosomes orother sequences having polymorphisms whose zygosity cases correlate withobservable phenotypes. See block 1304. In some embodiments, sequencinglibrary preparation allows the acquisition of the plurality of trainingsequence tags. In some embodiments, library preparation comprisesamplifying a plurality of training sequences before sequencing thecombined sample. In some embodiments, the plurality of trainingsequences comprises more than 10, 50, 60, 100, 500, 1000, or 5000sequences. Similar to the test-difference-value, thetraining-difference-values may also be obtained by different operationsand or normalized.

In some embodiments, process 1300 also involves obtaining distributionstatistics for the plurality of training-difference-values. See block1306. In some embodiments, the distribution statistics include a mixturemodel that describes the data as having two or more separate underlyingdistributions, each distribution corresponding to data associated with azygosity case. In some embodiments, the distribution statistics includethe central tendency (e.g., mean) and spread (e.g., standard deviation)of two or more underlying distributions.

In some embodiments, process 1300 also involves determining the zygosityof the sequence of interest for the fetus using thetest-difference-value and the distribution statistics for the pluralityof training-difference-values. See block 1308. In some embodiments, forinstance, if the test-difference-value falls within 1, 2, or 3 standarddeviations of the mean of one distribution that corresponding to afetal/maternal zygosity case, it is determined that the sequence ofinterest has such a zygosity case. Other criteria may be used, which canbe adjusted according to the empirical information obtained for thedistribution.

In some embodiments, instead of, or in addition to, examining thedifference value for one allele of a bi-allelic gene, the methodexamines the difference value for two alleles. The method involvesobtaining a difference of the difference value between two alleles. Thisdifference value between two alleles is obtained for the sequence ofinterest and the plurality of training sequences. The method alsoinvolves obtaining the distribution statistics for the difference valuebetween the two alleles for the training sequence, which is then used todetermine whether the sequence of interest belongs to a distributioncorresponding to a zygosity case as described above.

FIGS. 14A-14D illustrates a hypothetical example of the advantages ofsome embodiments involving analyses of mother-only cellular DNA overconventional method. FIG. 14A shows the distributions of minor allelecounts for three different zygosity cases: mother homozygous and fetusheterozygous (case 2), mother heterozygous and fetus homozygous (case3), and mother heterozygous and fetus heterozygous (case 4). Thedistribution for the zygosity case that both mother and fetus arehomozygous (case 1) is omitted because the minor allele should beabsent. It is difficult to differentiate cases 3 and 4 usingconventional methods, which discrimination is particularly important todetermine if the fetus is homozygous when the mother is a carrier of adisease related gene.

FIG. 14A shows hypothetical distributions of minor allele counts formultiple sequences, each sequence having two alleles and a total of 200sequence tags for the two alleles. The hypothetical data assumes a fetalfraction of 10%. For sequences in the mother heterozygous and fetushomozygous case (case 3), the expected mean of the minor allele count is90. For sequences in the mother heterozygous and fetus heterozygous case(case 4), the expected mean of the minor allele count is 100. Becausethe large spread and the small separation of the two distributions, fora test sequence with a 95 minor allele count it is difficult orimpossible to determine if the sequence belongs to one or the otherzygosity case. As such, some conventional methods consider both zygositycases “uninformative” for determining zygosity case. Such method canonly use data from the mother homozygous and fetus heterozygous case(case 2) to determine zygosity of the sequence. Fetal fraction (FF) maybe calculated after the zygosity case is determined as FF=2A/D for case2, wherein A is the minor allele count, and D is the total allelecounts.

Some conventional methods used both cellular DNA and cfDNA to determinefetal sequences. For instance, Bischoff et al. compared the efficacy ofassays using fetal cellular DNA and cfDNA for non-invasive prenatalscreening, and found that the cfDNA assay was at least four times moresensitive. Bischoff et al. (2002), Human Reproduction Update, Vol. 8,No. 6, pp. 439-500. Bischoff et al. thus concluded: “Cell-free fetal DNAmay be a more robust approach than intact cell analysis.” Moreover,Bischoff found no correction between the frequency of detecting fetalcellular DNA and the concentration of cfDNA. The lack of correlationargues against combining cellular DNA and cfDNA assays. As such,conventional methods did not provide means to combine both cellular DNAand cfDNA to achieve sensitivity and specificity even higher than usingcfDNA alone, or address case 3 above that is difficult to resolve usingconventional cfDNA methods. Some implementations described below combineboth cellular DNA and cfDNA to achieve advantages over conventionalmethods using cfDNA alone.

Some embodiments, such as method 1300, use the maternal cellular DNA tofactor out the noise in the cfDNA, thereby increasing the power todiscriminate case 3 from case 4. Using the hypothetical data of Table 1,also assuming 200 sequence tags for each training sequence, one maydeduce the cellular DNA and cfDNA allele counts for both alleles forheterozygous mother and homozygous fetus (case 3) and heterozygousmother and heterozygous fetus (case 4). Although the hypotheticalexamples illustrated here involve maternal cellular DNA, The processesdisclosed here may also be applied using fetal cellular DNA.

TABLE 2 Allele counts for allele a and b for case 3 (mother heterozygousand fetus homozygous) Cellular DNA cfDNA (maternal) (mixed) Differencevalues Allele a 104 95.6 Δa = −9.4 (cfDNA − cellular DNA) (minor) Alleleb 96 106.4 Δb = 10.4 (cfDNA − cellular DNA) (major) Δb − Δa = 19.8

TABLE 3 Allele counts for allele a and b for case 4 (mother and fetusheterozygous) Cellular DNA cfDNA (maternal) (mixed) Difference valuesAllele a 104 104 Δa = 0 (cfDNA − cellular DNA) Allele b 96 96 Δb = 0(cfDNA − cellular DNA) Δb − Δa = 0

Some embodiments, such as method 1300, use the maternal cellular DNA tofactor out the empirical noise in the cfDNA, thereby increasing thepower to discriminate case 3 from case 4. Using the hypothetical data ofTable 1, also assuming 200 sequence tags for each training sequence, onecan deduce the cellular DNA and cfDNA allele counts for both alleles forheterozygous mother and homozygous fetus (case 3) and heterozygousmother and heterozygous fetus (case 4).

Because the difference of allele counts cfDNA—cellular DNA is obtainedfrom sequencing libraries having the same empirical and processingconditions, it is expected that the variation common to both cfDNA andcellular DNA is removed by the subtraction (or other operations thatindicate the difference between the two libraries). This expectation,however, does not limit or determine the utility or patentability of themethod disclosed herein.

In some embodiments, the method involves obtaining the difference bysubtracting the sequence tag counts (or normalized counts) between thecfDNA versus the cellular DNA. Table 2 shows the difference value bysubtraction for case 3 data. Δa shows the difference value for allele a(minor allele), Δb shows the difference value for allele b (majorallele), and Δb−Δa shows the difference between Δb and Δa. Similarly,Table 3 shows the difference value by subtraction for case 4 data. Inthis example, only data from case 3 and case 4 are shown because thesetwo cases present challenge to conventional methods. In fact, theembodiments disclosed herein are also applicable to case 2 data.

FIGS. 14B-14C show the hypothetical distribution to the hypotheticaldata. FIG. 14B shows the distributions for Δb for case 3 and case 4.FIG. 14C shows the distributions for Δa for case 3 and case 4. FIG. 14Dshows the distributions for Δb-Δa for case 3 and case 4. Because thesubtraction of cellular DNA (labeled as gDNA in the figures) from thecfDNA removes empirical noise from the data, the spread of thedistributions are smaller as compared to the spread of the distributionof the minor allele count shown in FIG. 14A. This allows discriminationbetween the two distributions for the case 3 and case 4. Using a methoddisclosed herein, one may obtain distribution statistics of Δa, Δb, andΔb-Δa for training sequences. Using the distribution statistics and thedifference value for a sequence of interest, as disclosed above, one candetermine whether the sequence of interest belongs to case 3 or 4.Therefore, methods disclosed herein allow one to determine if a fetus ishomozygous or heterozygous when the mother is heterozygous. Given azygosity case, one may calculate fetal fraction (FF) using the allelecounts, such as determining FF=1−2A/D for case 3, wherein A is the minorallele count and D is the total count. Other methods known in the artalso may be used to calculate fetal fraction. In some embodiments, thiscalculation may adjust for the estimate of fetal fraction by consideringthe empirical bias observed in the maternal cellular DNA

In a conventional method using only cfDNA, the zygosity of the fetus isunknown. Therefore, only those SNPs for which the mother is homozygousand the fetus is heterozygous constitute “informative SNPs.” In thiscase, fetal fraction (FF) may be calculated as FF=2A/D, wherein A is theminor allele count, and D is the total allele counts.

In a conventional method using only cfDNA, SNPs where the mother may beheterozygous and the fetus is homozygous are not easily used withoutunderstanding the inherent “noise” in the heterozygous calls for themother. This is so because the minor allele frequency formother-hetero-fetus-homo zygosity case (case 3 above) is similar to thatfor mother-hetero-fetus-hetero case (case 4 above), both of which wereconsidered “uninformative” for determining FF.

In some embodiments involving analysis of fetal cellular DNA, bygenotyping fetal cellular DNA, the zygosity of a fetus may be obtainedusing data from fetal cellular DNA. This zygosity can help to determinefetal fraction (FF) of cfDNA using both “informative” and“uninformative” data. Fetal fraction may be calculated formother-hetero-fetus-homo zygosity case as FF=1−2A/D, wherein A is theminor allele count, and D is the total allele counts. The zygosity caseis ascertained by fetal zygosity determined from fetal cellular DNA andmaternal zygosity determined from allele frequencies of cfDNA.Therefore, some embodiments provide methods to obtain FF with highaccuracy by availing more data for the calculation.

Furthermore, this measure of FF allows a good estimate of CNV. If the FFof a particular sequence differs significantly from the normal FF, CNVmay be inferred for the sequence. For instance, if the fetal fraction is10%, when the mother is homozygous, and fetus heterozygous, then theminor allele frequency is expected to be 5%. However, if the fetus hasan extra copy of the sequence, then the minor allele frequency will be10%, which may be determined as significantly different from theexpected 5%.

The expected minor allele frequency may be obtained from trainingsequences obtained from the same sample, which training sequences aredifferent from the sequence of interest being tested. Therefore, someembodiments of the disclosure involve processing and analyzing aplurality of training sequences. In some embodiments, the cellular DNAand cfDNA are enriched for training sequences or sites. In someembodiments, more than about 10, 50, 60, 100, 500, 1000, or 5000 SNPsites are used as training sites to understand distribution counts ofSNPs sequence tags in the assay where the mother is heterozygous and thefetus is homozygous. In some embodiments, when a sequence of interesthas a minor allele frequency different from the training sequence by acriterion, a CNV call is determined.

The CNV call uses a first sequence from the fetal cellular DNA todetermine the zygosity of the fetus. The fetal cfDNA may also providethe CNV for the first sequence. However, the same method may also beapplied to obtain the CNV for a second sequence, which may not beavailable from the fetal cellular DNA due to the scarcity of the fetalcellular DNA. Therefore, the disclosed methods combining cellular DNAand cfDNA provide an advantage relative to using cellular DNA alone.

Evaluating Copy Number Variation (CNV)

Particularly useful methods for evaluating CNV using mother-and-fetuscfDNA are further described below. Some embodiments of this disclosureprovide methods using both mother cellular DNA and mother-and-fetuscfDNA for validation of the CNV obtained from cfDNA alone, boosting theconfidence of CNV evaluation. Some embodiments use both fetus cellularDNA and mother-and-fetus cfDNA, for example, as a mixture in multiplexdetection formats, to evaluate CNV. Some embodiments provide methods fordirectly calculating CNV using both cellular DNA and cfDNA.

Using sequence coverage values for mother-and-fetus cfDNAs, e.g.,according to the methods described below, one can determine copy numberand CNV of sequences, chromosomes, or chromosome segments. In someembodiments, the method for determining the presence or absence of anycomplete fetal chromosomal aneuploidies using cfDNA from a maternal testsample comprises (a) obtaining sequence information for fetal andmaternal nucleic acids in the maternal test sample; (b) using thesequence information and the method described herein to identify anumber of sequence tags or sequence coverage quantity derived therefromfor each of the chromosomes of interest selected from chromosomes 1-22,X and Y and to identify a number of sequence tags for one or morenormalizing chromosome sequences; (c) using the number of sequence tagsidentified for each of the chromosomes of interest and the number ofsequence tags identified for each of the normalizing chromosomes tocalculate a single chromosome dose for each of the chromosomes ofinterests; and (d) comparing each chromosome dose to a threshold value,and thereby determining the presence or absence of any complete fetalchromosomal aneuploidies in the maternal test sample.

In some embodiments, step (a) described above can comprise sequencing atleast a portion of the nucleic acid molecules of a test sample to obtainsaid sequence information for the fetal and maternal nucleic acidmolecules of the test sample. In some embodiments, step (c) comprisescalculating a single chromosome dose for each of the chromosomes ofinterest as the ratio of the number of sequence tags identified for eachof the chromosomes of interest and the number of sequence tagsidentified for the normalizing chromosome sequence(s), where anormalizing sequence is a robust chromosome that is unlikely to beaneuploid. In some other embodiments, chromosome dose is based onprocessed sequence coverage quantities derived from the number ofsequence tags. In some embodiments, only unique, non-redundant sequencetags are used to calculate the processed sequence coverage quantities.In some embodiments, the processed sequence coverage quantity is asequence tag density ratio, which is the number of sequence tagsstandardized by sequence length. In some embodiments, the processedsequence coverage quantity is a normalized sequence tag, which is thenumber of sequence tags of a sequence of interest divided by all or asubstantial portion of the genome.

In some embodiments, a chromosome dose is calculated as the ratio of theprocessed sequence coverage quantities for each of the chromosomes ofinterest and processed sequence coverage quantities for the normalizingchromosome sequence(s).

In any one of the embodiments above, one or more steps of the method arerepeated for test samples from different maternal subjects. In any oneof the embodiments above, the method can further comprise calculating anormalized chromosome value (NCV), wherein the NCV relates thechromosome dose to the mean of the corresponding chromosome dose in aset of qualified samples as:

${NCV}_{ij} = \frac{x_{ij} - {\hat{\mu}}_{j}}{{\hat{\sigma}}_{j}}$where {circumflex over (μ)}_(j) and {circumflex over (ν)}_(j) are theestimated mean and standard deviation, respectively, for the j-thchromosome dose in a set of qualified samples, and x_(ij) is theobserved j-th chromosome dose for test sample i.

In some embodiments, NCV can be calculated “on the fly” by relating thechromosome dose of a chromosome of interest in a test sample to themedian of the corresponding chromosome dose in multiplexed samplessequenced on the same flow cells as:

${NCV}_{ij} = \frac{x_{ij} - M_{j}}{{\hat{\sigma}}_{j}}$where M_(j) is the estimated median for the j-th chromosome dose in aset of multiplexed samples sequenced on the same flow cell; {circumflexover (σ)}_(j) is the standard deviation for the j-th chromosome dose inone or more sets of multiplexed samples sequenced on one or more flowcells, and x_(i) is the observed j-th chromosome dose for test sample i.In this embodiment, test sample i is one of the multiplexed samplessequenced on the same flow cell from which M_(j) is determined.

In some embodiments, a method is provided for determining the presenceor absence of different partial fetal chromosomal aneuploidies in amaternal test sample comprising fetal and maternal nucleic acids. Themethod involves procedures analogous to the method for detectingcomplete aneuploidy as outlined above. However, instead of analyzing acomplete chromosome, a segment of a chromosome is analyzed. Accordingly,instead of a NCV, a normalized segment value (NSV) is obtained forevaluation of the chromosome segment. See US Patent ApplicationPublication No. 2013/0029852, and US Patent Application No. 61/893,830,which are incorporated by reference.

In some embodiments, the determination of CNV comprises calculating aNCV or NSV that relates the chromosome or segment dose to the mean ofthe corresponding chromosome or segment dose in a set of qualifiedsamples as described above. Then CNV can be determined by comparing theNCV/NSV to a predetermined copy number evaluatiom threshold value.

In some embodiments, the FF for a chromosome can be calculated accordingto the following equation.

${FF}_{ij} = {{2 \times {\frac{{NCV}_{ij} \times {\hat{\sigma}}_{j}}{{\hat{\mu}}_{j}}}} = {2 \times {NCV} \times {CV}}}$

Namely, for every NCV of a chromosome of interest, an expected fetalfraction associated with the given NCV value can be calculated from theCV based on the mean and standard deviation of the chromosome ratio forthe chromosome of interest across a population of unaffected samples.

Some embodiments of the disclosure provide a method for validating theNCV obtained using only cfDNA. For instance, a plurality of estimates ofFF from various zygosity cases obtained using both mixed cfDNA andmaternal cellular DNA as described elsewhere herein may be combined toobtain distribution statistics of FF. Then the FF obtained using onlycfDNA can be compared to the FF distribution using both cfDNA andcellular DNA. If the FF obtained with cfDNA falls outside a decisioncriterion, the NCV obtained using cfDNA only may need to be retested forexperimental error or mosaicism.

Some embodiments of the disclosure provide methods for directlydetermining CNV using both cfDNA and maternal cellular DNA, which CNVestimate has a higher sensitivity and lower noise than using cfDNAalone. In these embodiments, the methods take advantage of the FFobtained using both the cellular DNA and cfDNA as described above toreduce noise in the estimate. In some embodiments, a chromosome dose ofsequence j is calculated as:x _(j) =m _(j)−average(n _(i))×(1−FF)wherein m_(j) is a standardized coverage of sequence j standardized bysequence length obtained using cfDNA data, n_(i) is a standardizedcoverage of a normalizing sequence i using cfDNA data, and FF is thefetal fraction obtained using both cellular DNA and cfDNA as describedabove. This chromosome dose of sequence j has the maternal DNA coveragesubtracted from the data. Then NCV may be calculated as described asdescribed above:

${NCV}_{ij} = \frac{x_{ij} - {\hat{\mu}}_{j}}{{\hat{\sigma}}_{j}}$

Here {circumflex over (μ)}_(j) is the chromosome dose for controlpregnant women for sequence j calculated in the same way as x_(j). Thechromosome dose of sequence j in this method has the maternal DNAcoverage subtracted from the data, thereby decreasing the baseline ofcomparison and increasing the signal to noise ratio of the NCV estimate.Then a CNV call may be based on this more accurate NCV compared tomethod using cfDNA only. Other embodiments may use different methods tocombine the data from both cfDNA and cellular DNA to improve the signaland reduce the noise for CNV calls.

Marker Nucleic Acids for Tracking and Verifying Sample Integrity

In various embodiments verification of the integrity of the samples andsample tracking can be accomplished by sequencing mixtures of samplegenomic nucleic acids, e.g., cfDNA, and accompanying marker nucleicacids that have been introduced into the samples, e.g., prior toprocessing.

Marker nucleic acids can be combined with the test sample (e.g.,biological source sample) and subjected to processes that include, forexample, one or more of the steps of fractionating the biological sourcesample, e.g., obtaining an essentially cell-free plasma fraction from awhole blood sample, purifying nucleic acids from a fractionated, e.g.,plasma, or unfractionated biological source sample, e.g., a tissuesample, and sequencing. In some embodiments, sequencing comprisespreparing a sequencing library. The sequence or combination of sequencesof the marker molecules that are combined with a source sample is chosento be unique to the source sample. In some embodiments, the uniquemarker molecules in a sample all have the same sequence. In otherembodiments, the unique marker molecules in a sample are a plurality ofsequences, e.g., a combination of two, three, four, five, six, seven,eight, nine, ten, fifteen, twenty, or more different sequences.

In one embodiment, the integrity of a sample can be verified using aplurality of marker nucleic acid molecules having identical sequences.Alternatively, the identity of a sample can be verified using aplurality of marker nucleic acid molecules that have at least two, atleast three, at least four, at least five, at least six, at least seven,at least eight, at least nine, at least ten, at least 11, at least 12,at least 13, at least 14, at least 15, at least 16, at least 17m, atleast 18, at least 19, at least 20, at least 25, at least 30, at least35, at least 40, at least 50, or more different sequences. Verificationof the integrity of the plurality of biological samples, i.e., two ormore biological samples, requires that each of the two or more samplesbe marked with marker nucleic acids that have sequences that are uniqueto each of the plurality of test sample that is being marked. Forexample, a first sample can be marked with a marker nucleic acid havingsequence A, and a second sample can be marked with a marker nucleic acidhaving sequence B. Alternatively, a first sample can be marked withmarker nucleic acid molecules all having sequence A, and a second samplecan be marked with a mixture of sequences B and C, wherein sequences A,B and C are marker molecules having different sequences.

The marker nucleic acid(s) can be added to the sample at any stage ofsample preparation that occurs prior to library preparation (iflibraries are to be prepared) and sequencing. In one embodiment, markermolecules can be combined with an unprocessed source sample. Forexample, the marker nucleic acid can be provided in a collection tubethat is used to collect a blood sample. Alternatively, the markernucleic acids can be added to the blood sample following the blood draw.In one embodiment, the marker nucleic acid is added to the vessel thatis used to collect a biological fluid sample, e.g., the marker nucleicacid(s) are added to a blood collection tube that is used to collect ablood sample. In another embodiment, the marker nucleic acid(s) areadded to a fraction of the biological fluid sample. For example, themarker nucleic acid is added to the plasma and/or serum fraction of ablood sample, e.g., a maternal plasma sample. In yet another embodiment,the marker molecules are added to a purified sample, e.g., a sample ofnucleic acids that have been purified from a biological sample. Forexample, the marker nucleic acid is added to a sample of purifiedmaternal and fetal cfDNA. Similarly, the marker nucleic acids can beadded to a biopsy specimen prior to processing the specimen. In someembodiments, the marker nucleic acids can be combined with a carrierthat delivers the marker molecules into the cells of the biologicalsample. Cell-delivery carriers include pH-sensitive and cationicliposomes.

In various embodiments, the marker molecules have antigenomic sequences,that are sequences that are absent from the genome of the biologicalsource sample. In an exemplary embodiment, the marker molecules that areused to verify the integrity of a human biological source sample havesequences that are absent from the human genome. In an alternativeembodiment, the marker molecules have sequences that are absent from thesource sample and from any one or more other known genomes. For example,the marker molecules that are used to verify the integrity of a humanbiological source sample have sequences that are absent from the humangenome and from the mouse genome. The alternative allows for verifyingthe integrity of a test sample that comprises two or more genomes. Forexample, the integrity of a human cell-free DNA sample obtained from asubject affected by a pathogen, e.g., a bacterium, can be verified usingmarker molecules having sequences that are absent from both the humangenome and the genome of the affecting bacterium. Sequences of genomesof numerous pathogens, e.g., bacteria, viruses, yeasts, fungi, protozoaetc., are publicly available on the World Wide Web atncbi.nlm.nih.gov/genomes. In another embodiment, marker molecules arenucleic acids that have sequences that are absent from any known genome.The sequences of marker molecules can be randomly generatedalgorithmically.

In various embodiments the marker molecules can be naturally-occurringdeoxyribonucleic acids (DNA), ribonucleic acids or artificial nucleicacid analogs (nucleic acid mimics) including peptide nucleic acids(PMA), morpholino nucleic acid, locked nucleic acids, glycol nucleicacids, and threose nucleic acids, which are distinguished fromnaturally-occurring DNA or RNA by changes to the backbone of themolecule or DNA mimics that do not have a phosphodiester backbone. Thedeoxyribonucleic acids can be from naturally-occurring genomes or can begenerated in a laboratory through the use of enzymes or by solid phasechemical synthesis. Chemical methods can also be used to generate theDNA mimics that are not found in nature. Derivatives of DNA are that areavailable in which the phosphodiester linkage has been replaced but inwhich the deoxyribose is retained include but are not limited to DNAmimics having backbones formed by thioformacetal or a carboxamidelinkage, which have been shown to be good structural DNA mimics. OtherDNA mimics include morpholino derivatives and the peptide nucleic acids(PNA), which contain an N-(2-aminoethyl)glycine-based pseudopeptidebackbone (Ann Rev Biophys Biomol Struct 24:167-183 [1995]). PNA is anextremely good structural mimic of DNA (or of ribonucleic acid [RNA]),and PNA oligomers are able to form very stable duplex structures withWatson-Crick complementary DNA and RNA (or PNA) oligomers, and they canalso bind to targets in duplex DNA by helix invasion (Mol Biotechnol26:233-248 [2004]. Another good structural mimic/analog of DNA analogthat can be used as a marker molecule is phosphorothioate DNA in whichone of the non-bridging oxygens is replaced by a sulfur. Thismodification reduces the action of endo-and exonucleases2 including 5′to 3′ and 3′ to 5′ DNA POL 1 exonuclease, nucleases S1 and P1, RNases,serum nucleases and snake venom phosphodiesterase.

The length of the marker molecules can be distinct or indistinct fromthat of the sample nucleic acids, i.e., the length of the markermolecules can be similar to that of the sample genomic molecules, or itcan be greater or smaller than that of the sample genomic molecules. Thelength of the marker molecules is measured by the number of nucleotideor nucleotide analog bases that constitute the marker molecule. Markermolecules having lengths that differ from those of the sample genomicmolecules can be distinguished from source nucleic acids usingseparation methods known in the art. For example, differences in thelength of the marker and sample nucleic acid molecules can be determinedby electrophoretic separation, e.g., capillary electrophoresis. Sizedifferentiation can be advantageous for quantifying and assessing thequality of the marker and sample nucleic acids. Preferably, the markernucleic acids are shorter than the genomic nucleic acids, and ofsufficient length to exclude them from being mapped to the genome of thesample. For example, as a 30 base human sequence is needed to uniquelymap it to a human genome. Accordingly in certain embodiments, markermolecules used in sequencing bioassays of human samples should be atleast 30 bp in length.

The choice of length of the marker molecule is determined primarily bythe sequencing technology that is used to verify the integrity of asource sample. The length of the sample genomic nucleic acids beingsequenced can also be considered. For example, some sequencingtechnologies employ clonal amplification of polynucleotides, which canrequire that the genomic polynucleotides that are to be clonallyamplified be of a minimum length. For example, sequencing using theIllumina GAII sequence analyzer includes an in vitro clonalamplification by bridge PCR (also known as cluster amplification) ofpolynucleotides that have a minimum length of 110 bp, to which adaptorsare ligated to provide a nucleic acid of at least 200 bp and less than600 bp that can be clonally amplified and sequenced. In someembodiments, the length of the adaptor-ligated marker molecule isbetween about 200 bp and about 600 bp, between about 250 bp and 550 bp,between about 300 bp and 500 bp, or between about 350 and 450. In otherembodiments, the length of the adaptor-ligated marker molecule is about200 bp. For example, when sequencing fetal cfDNA that is present in amaternal sample, the length of the marker molecule can be chosen to besimilar to that of fetal cfDNA molecules. Thus, in one embodiment, thelength of the marker molecule used in an assay that comprises massivelyparallel sequencing of cfDNA in a maternal sample to determine thepresence or absence of a fetal chromosomal aneuploidy, can be about 150bp, about 160 bp, 170 bp, about 180 bp, about 190 bp or about 200 bp;preferably, the marker molecule is about 170 pp. Other sequencingapproaches, e.g., SOLiD sequencing, Polony Sequencing and 454 sequencinguse emulsion PCR to clonally amplify DNA molecules for sequencing, andeach technology dictates the minimum and the maximum length of themolecules that are to be amplified. The length of marker molecules to besequenced as clonally amplified nucleic acids can be up to about 600 bp.In some embodiments, the length of marker molecules to be sequenced canbe greater than 600 bp.

Single molecule sequencing technologies, that do not employ clonalamplification of molecules, and are capable of sequencing nucleic acidsover a very broad range of template lengths, in most situations do notrequire that the molecules to be sequenced be of any specific length.However, the yield of sequences per unit mass is dependent on the numberof 3′ end hydroxyl groups, and thus having relatively short templatesfor sequencing is more efficient than having long templates. If startingwith nucleic acids longer than 1000 nt, it is generally advisable toshear the nucleic acids to an average length of 100 to 200 nt so thatmore sequence information can be generated from the same mass of nucleicacids. Thus, the length of the marker molecule can range from tens ofbases to thousands of bases. The length of marker molecules used forsingle molecule sequencing can be up to about 25 bp, up to about 50 bp,up to about 75 bp, up to about 100 bp, up to about 200 bp, up to about300 bp, up to about 400 bp, up to about 500 bp, up to about 600 bp, upto about 700 bp, up to about 800 bp, up to about 900 bp, up to about1000 bp, or more in length.

The length chosen for a marker molecule is also determined by the lengthof the genomic nucleic acid that is being sequenced. For example, cfDNAcirculates in the human bloodstream as genomic fragments of cellulargenomic DNA. Fetal cfDNA molecules found in the plasma of pregnant womenare generally shorter than maternal cfDNA molecules (Chan et al., ClinChem 50:8892 [2004]). Size fractionation of circulating fetal DNA hasconfirmed that the average length of circulating fetal DNA fragments is<300 bp, while maternal DNA has been estimated to be between about 0.5and 1 Kb (Li et al., Clin Chem, 50: 1002-1011 [2004]). These findingsare consistent with those of Fan et al., who determined using NGS thatfetal cfDNA is rarely >340 bp (Fan et al., Clin Chem 56:1279-1286[2010]). DNA isolated from urine with a standard silica-based methodconsists of two fractions, high molecular weight DNA, which originatesfrom shed cells and low molecular weight (150-250 base pair) fraction oftransrenal DNA (Tr-DNA) (Botezatu et al., Clin Chem. 46: 1078-1084,2000; and Su et al., J Mol. Diagn. 6: 101-107, 2004). The application ofnewly developed technique for isolation of cell-free nucleic acids frombody fluids to the isolation of transrenal nucleic acids has revealedthe presence in urine of DNA and RNA fragments much shorter than 150base pairs (U.S. Patent Application Publication No. 20080139801). Inembodiments, wherein cfDNA is the genomic nucleic acid that issequenced, marker molecules that are chosen can be up to about thelength of the cfDNA. For example, the length of marker molecules used inmaternal cfDNA samples to be sequenced as single nucleic acid moleculesor as clonally amplified nucleic acids can be between about 100 bp and600. In other embodiments, the sample genomic nucleic acids arefragments of larger molecules. For example, a sample genomic nucleicacid that is sequenced is fragmented cellular DNA. In embodiments, whenfragmented cellular DNA is sequenced, the length of the marker moleculescan be up to the length of the DNA fragments. In some embodiments, thelength of the marker molecules is at least the minimum length requiredfor mapping the sequence read uniquely to the appropriate referencegenome. In other embodiments, the length of the marker molecule is theminimum length that is required to exclude the marker molecule frombeing mapped to the sample reference genome.

In addition, marker molecules can be used to verify samples that are notassayed by nucleic acid sequencing, and that can be verified by commonbio-techniques other than sequencing, e.g., real-time PCR.

Sample Controls (e.g., in Process Positive Controls for Sequencingand/or Analysis).

In various embodiments marker sequences introduced into the samples,e.g., as described above, can function as positive controls to verifythe accuracy and efficacy of sequencing and subsequent processing andanalysis.

Accordingly, compositions and method for providing an in-processpositive control (IPC) for sequencing DNA in a sample are provided. Incertain embodiments, positive controls are provided for sequencing cfDNAin a sample comprising a mixture of genomes are provided. An IPC can beused to relate baseline shifts in sequence information obtained fromdifferent sets of samples, e.g., samples that are sequenced at differenttimes on different sequencing runs. Thus, for example, an IPC can relatethe sequence information obtained for a maternal test sample to thesequence information obtained from a set of qualified samples that weresequenced at a different time.

Similarly, in the case of segment analysis, an IPC can relate thesequence information obtained from a subject for particular segment(s)to the sequence obtained from a set of qualified samples (of similarsequences) that were sequenced at a different time. In certainembodiments an IPC can relate the sequence information obtained from asubject for particular cancer-related loci to the sequence informationobtained from a set of qualified samples (e.g., from a knownamplification/deletion, and the like).

In addition, IPCs can be used as markers to track sample(s) through thesequencing process. IPCs can also provide a qualitative positivesequence dose value, e.g., NCV, for one or more aneuploidies ofchromosomes of interest, e.g., trisomy 21, trisomy 13, trisomy 18 toprovide proper interpretation, and to ensure the dependability andaccuracy of the data. In certain embodiments IPCs can be created tocomprise nucleic acids from male and female genomes to provide doses forchromosomes X and Y in a maternal sample to determine whether the fetusis male.

The type and the number of in-process controls depends on the type ornature of the test needed. For example, for a test requiring thesequencing of DNA from a sample comprising a mixture of genomes todetermine whether a chromosomal aneuploidy exists, the in-processcontrol can comprise DNA obtained from a sample known comprising thesame chromosomal aneuploidy that is being tested. In some embodiments,the IPC includes DNA from a sample known to comprise an aneuploidy of achromosome of interest. For example, the IPC for a test to determine thepresence or absence of a fetal trisomy, e.g., trisomy 21, in a maternalsample comprises DNA obtained from an individual with trisomy 21. Insome embodiments, the IPC comprises a mixture of DNA obtained from twoor more individuals with different aneuploidies. For example, for a testto determine the presence or absence of trisomy 13, trisomy 18, trisomy21, and monosomy X, the IPC comprises a combination of DNA samplesobtained from pregnant women each carrying a fetus with one of thetrisomies being tested. In addition to complete chromosomalaneuploidies, IPCs can be created to provide positive controls for teststo determine the presence or absence of partial aneuploidies.

An IPC that serves as the control for detecting a single aneuploidy canbe created using a mixture of cellular genomic DNA obtained from a twosubjects one being the contributor of the aneuploid genome. For example,an IPC that is created as a control for a test to determine a fetaltrisomy, e.g., trisomy 21, can be created by combining genomic DNA froma male or female subject carrying the trisomic chromosome with genomicDNA with a female subject known not to carry the trisomic chromosome.Genomic DNA can be extracted from cells of both subjects, and sheared toprovide fragments of between about 100-400 bp, between about 150-350 bp,or between about 200-300 bp to simulate the circulating cfDNA fragmentsin maternal samples. The proportion of fragmented DNA from the subjectcarrying the aneuploidy, e.g., trisomy 21, is chosen to simulate theproportion of circulating fetal cfDNA found in maternal samples toprovide an IPC comprising a mixture of fragmented DNA comprising about5%, about 10%, about 15%, about 20%, about 25%, about 30%, of DNA fromthe subject carrying the aneuploidy. The IPC can comprise DNA fromdifferent subjects each carrying a different aneuploidy. For example,the IPC can comprise about 80% of the unaffected female DNA, and theremaining 20% can be DNA from three different subjects each carrying atrisomic chromosome 21, a trisomic chromosome 13, and a trisomicchromosome 18. The mixture of fragmented DNA is prepared for sequencing.Processing of the mixture of fragmented DNA can comprise preparing asequencing library, which can be sequenced using any massively parallelmethods in singleplex or multiplex fashion. Stock solutions of thegenomic IPC can be stored and used in multiple diagnostic tests.

Alternatively the IPC can be created using cfDNA obtained from a motherknown to carry a fetus with a known chromosomal aneuploidy. For example,cfDNA can be obtained from a pregnant woman carrying a fetus withtrisomy 21. The cfDNA is extracted from the maternal sample, and clonedinto a bacterial vector and grown in bacteria to provide an ongoingsource of the IPC. The DNA can be extracted from the bacterial vectorusing restriction enzymes. Alternatively, the cloned cfDNA can beamplified by, e.g., PCR. The IPC DNA can be processed for sequencing inthe same runs as the cfDNA from the test samples that are to be analyzedfor the presence or absence of chromosomal aneuploidies.

While the creation of IPCs is described above with respect to trisomies,it will be appreciated that IPCs can be created to reflect other partialaneuploidies including for example, various segment amplification and/ordeletions. Thus, for example, where various cancers are known to beassociated with particular amplifications (e.g., breast cancerassociated with 20Q13) IPCs can be created that incorporate those knownamplifications.

Sequencing Methods

As indicated above, the prepared samples (e.g., Sequencing Libraries)are sequenced as part of the procedure for determining a sequence ofinterest and for evaluating copy number variation(s). Any of a number ofsequencing technologies can be utilized.

Some sequencing technologies are available commercially, such as thesequencing-by-hybridization platform from Affymetrix Inc. (Sunnyvale,Calif.) and the sequencing-by-synthesis platforms from 454 Life Sciences(Bradford, Conn.), Illumina/Solexa (Hayward, Calif.) and HelicosBiosciences (Cambridge, Mass.), and the sequencing-by-ligation platformfrom Applied Biosystems (Foster City, Calif.), as described below. Inaddition to the single molecule sequencing performed usingsequencing-by-synthesis of Helicos Biosciences, other single moleculesequencing technologies include, but are not limited to, the SMRT™technology of Pacific Biosciences, the ION TORRENT™ technology, andnanopore sequencing developed for example, by Oxford NanoporeTechnologies.

While the automated Sanger method is considered as a ‘first generation’technology, Sanger sequencing including the automated Sanger sequencing,can also be employed in the methods described herein. Additionalsuitable sequencing methods include, but are not limited to nucleic acidimaging technologies, e.g., atomic force microscopy (AFM) ortransmission electron microscopy (TEM). Illustrative sequencingtechnologies are described in greater detail below.

In one illustrative, but non-limiting, embodiment, the methods describedherein comprise obtaining sequence information for the nucleic acids ina test sample, e.g., cfDNA in a maternal sample, cfDNA or cellular DNAin a subject being screened for a cancer, and the like, using singlemolecule sequencing technology of the Helicos True Single MoleculeSequencing (tSMS) technology (e.g. as described in Harris T. D. et al.,Science 320:106-109 [2008]). In the tSMS technique, a DNA sample iscleaved into strands of approximately 100 to 200 nucleotides, and apolyA sequence is added to the 3′ end of each DNA strand. Each strand islabeled by the addition of a fluorescently labeled adenosine nucleotide.The DNA strands are then hybridized to a flow cell, which containsmillions of oligo-T capture sites that are immobilized to the flow cellsurface. In certain embodiments the templates can be at a density ofabout 100 million templates/cm2. The flow cell is then loaded into aninstrument, e.g., HeliScope™ sequencer, and a laser illuminates thesurface of the flow cell, revealing the position of each template. A CCDcamera can map the position of the templates on the flow cell surface.The template fluorescent label is then cleaved and washed away. Thesequencing reaction begins by introducing a DNA polymerase and afluorescently labeled nucleotide. The oligo-T nucleic acid serves as aprimer. The polymerase incorporates the labeled nucleotides to theprimer in a template directed manner. The polymerase and unincorporatednucleotides are removed. The templates that have directed incorporationof the fluorescently labeled nucleotide are discerned by imaging theflow cell surface. After imaging, a cleavage step removes thefluorescent label, and the process is repeated with other fluorescentlylabeled nucleotides until the desired read length is achieved. Sequenceinformation is collected with each nucleotide addition step. Wholegenome sequencing by single molecule sequencing technologies excludes ortypically obviates PCR-based amplification in the preparation of thesequencing libraries, and the methods allow for direct measurement ofthe sample, rather than measurement of copies of that sample.

In another illustrative, but non-limiting embodiment, the methodsdescribed herein comprise obtaining sequence information for the nucleicacids in the test sample, e.g., cfDNA in a maternal test sample, cfDNAor cellular DNA in a subject being screened for a cancer, and the like,using the 454 sequencing (Roche) (e.g. as described in Margulies, M. etal. Nature 437:376-380 [2005]). 454 sequencing typically involves twosteps. In the first step, DNA is sheared into fragments of approximately300-800 base pairs, and the fragments are blunt-ended. Oligonucleotideadaptors are then ligated to the ends of the fragments. The adaptorsserve as primers for amplification and sequencing of the fragments. Thefragments can be attached to DNA capture beads, e.g.,streptavidin-coated beads using, e.g., Adaptor B, which contains5′-biotin tag. The fragments attached to the beads are PCR amplifiedwithin droplets of an oil-water emulsion. The result is multiple copiesof clonally amplified DNA fragments on each bead. In the second step,the beads are captured in wells (e.g., picoliter-sized wells).Pyrosequencing is performed on each DNA fragment in parallel. Additionof one or more nucleotides generates a light signal that is recorded bya CCD camera in a sequencing instrument. The signal strength isproportional to the number of nucleotides incorporated. Pyrosequencingmakes use of pyrophosphate (PPi) which is released upon nucleotideaddition. PPi is converted to ATP by ATP sulfurylase in the presence ofadenosine 5′ phosphosulfate. Luciferase uses ATP to convert luciferin tooxyluciferin, and this reaction generates light that is measured andanalyzed.

In another illustrative, but non-limiting, embodiment, the methodsdescribed herein comprises obtaining sequence information for thenucleic acids in the test sample, e.g., cfDNA in a maternal test sample,cfDNA or cellular DNA in a subject being screened for a cancer, and thelike, using the SOLiD™ technology (Applied Biosystems). In SOLiD™sequencing-by-ligation, genomic DNA is sheared into fragments, andadaptors are attached to the 5′ and 3′ ends of the fragments to generatea fragment library. Alternatively, internal adaptors can be introducedby ligating adaptors to the 5′ and 3′ ends of the fragments,circularizing the fragments, digesting the circularized fragment togenerate an internal adaptor, and attaching adaptors to the 5′ and 3′ends of the resulting fragments to generate a mate-paired library. Next,clonal bead populations are prepared in microreactors containing beads,primers, template, and PCR components. Following PCR, the templates aredenatured and beads are enriched to separate the beads with extendedtemplates. Templates on the selected beads are subjected to a 3′modification that permits bonding to a glass slide. The sequence can bedetermined by sequential hybridization and ligation of partially randomoligonucleotides with a central determined base (or pair of bases) thatis identified by a specific fluorophore. After a color is recorded, theligated oligonucleotide is cleaved and removed and the process is thenrepeated.

In another illustrative, but non-limiting, embodiment, the methodsdescribed herein comprise obtaining sequence information for the nucleicacids in the test sample, e.g., cfDNA in a maternal test sample, cfDNAor cellular DNA in a subject being screened for a cancer, and the like,using the single molecule, real-time (SMRT™) sequencing technology ofPacific Biosciences. In SMRT sequencing, the continuous incorporation ofdye-labeled nucleotides is imaged during DNA synthesis. Single DNApolymerase molecules are attached to the bottom surface of individualzero-mode wavelength detectors (ZMW detectors) that obtain sequenceinformation while phospholinked nucleotides are being incorporated intothe growing primer strand. A ZMW detector comprises a confinementstructure that enables observation of incorporation of a singlenucleotide by DNA polymerase against a background of fluorescentnucleotides that rapidly diffuse in an out of the ZMW (e.g., inmicroseconds). It typically takes several milliseconds to incorporate anucleotide into a growing strand. During this time, the fluorescentlabel is excited and produces a fluorescent signal, and the fluorescenttag is cleaved off. Measurement of the corresponding fluorescence of thedye indicates which base was incorporated. The process is repeated toprovide a sequence.

In another illustrative, but non-limiting embodiment, the methodsdescribed herein comprise obtaining sequence information for the nucleicacids in the test sample, e.g., cfDNA in a maternal test sample, cfDNAor cellular DNA in a subject being screened for a cancer, and the like,using nanopore sequencing (e.g. as described in Soni G V and Meller A.Clin Chem 53: 1996-2001 [2007]). Nanopore sequencing DNA analysistechniques are developed by a number of companies, including, forexample, Oxford Nanopore Technologies (Oxford, United Kingdom),Sequenom, NABsys, and the like. Nanopore sequencing is a single-moleculesequencing technology whereby a single molecule of DNA is sequenceddirectly as it passes through a nanopore. A nanopore is a small hole,typically of the order of 1 nanometer in diameter. Immersion of ananopore in a conducting fluid and application of a potential (voltage)across it results in a slight electrical current due to conduction ofions through the nanopore. The amount of current that flows is sensitiveto the size and shape of the nanopore. As a DNA molecule passes througha nanopore, each nucleotide on the DNA molecule obstructs the nanoporeto a different degree, changing the magnitude of the current through thenanopore in different degrees. Thus, this change in the current as theDNA molecule passes through the nanopore provides a read of the DNAsequence.

In another illustrative, but non-limiting, embodiment, the methodsdescribed herein comprises obtaining sequence information for thenucleic acids in the test sample, e.g., cfDNA in a maternal test sample,cfDNA or cellular DNA in a subject being screened for a cancer, and thelike, using the chemical-sensitive field effect transistor (chemFET)array (e.g., as described in U.S. Patent Application Publication No.2009/0026082). In one example of this technique, DNA molecules can beplaced into reaction chambers, and the template molecules can behybridized to a sequencing primer bound to a polymerase. Incorporationof one or more triphosphates into a new nucleic acid strand at the 3′end of the sequencing primer can be discerned as a change in current bya chemFET. An array can have multiple chemFET sensors. In anotherexample, single nucleic acids can be attached to beads, and the nucleicacids can be amplified on the bead, and the individual beads can betransferred to individual reaction chambers on a chemFET array, witheach chamber having a chemFET sensor, and the nucleic acids can besequenced.

In another embodiment, the present method comprises obtaining sequenceinformation for the nucleic acids in the test sample, e.g., cfDNA in amaternal test sample, using the Halcyon Molecular's technology, whichuses transmission electron microscopy (TEM). The method, termedIndividual Molecule Placement Rapid Nano Transfer (IMPRNT), comprisesutilizing single atom resolution transmission electron microscopeimaging of high-molecular weight (150 kb or greater) DNA selectivelylabeled with heavy atom markers and arranging these molecules onultra-thin films in ultra-dense (3 nm strand-to-strand) parallel arrayswith consistent base-to-base spacing. The electron microscope is used toimage the molecules on the films to determine the position of the heavyatom markers and to extract base sequence information from the DNA. Themethod is further described in PCT patent publication WO 2009/046445.The method allows for sequencing complete human genomes in less than tenminutes.

In another embodiment, the DNA sequencing technology is the Ion Torrentsingle molecule sequencing, which pairs semiconductor technology with asimple sequencing chemistry to directly translate chemically encodedinformation (A, C, G, T) into digital information (0, 1) on asemiconductor chip. In nature, when a nucleotide is incorporated into astrand of DNA by a polymerase, a hydrogen ion is released as abyproduct. Ion Torrent uses a high-density array of micro-machined wellsto perform this biochemical process in a massively parallel way. Eachwell holds a different DNA molecule. Beneath the wells is anion-sensitive layer and beneath that an ion sensor. When a nucleotide,for example a C, is added to a DNA template and is then incorporatedinto a strand of DNA, a hydrogen ion will be released. The charge fromthat ion will change the pH of the solution, which can be detected byIon Torrent's ion sensor. The sequencer—essentially the world's smallestsolid-state pH meter—calls the base, going directly from chemicalinformation to digital information. The Ion personal Genome Machine(PGM™) sequencer then sequentially floods the chip with one nucleotideafter another. If the next nucleotide that floods the chip is not amatch. No voltage change will be recorded and no base will be called. Ifthere are two identical bases on the DNA strand, the voltage will bedouble, and the chip will record two identical bases called. Directdetection allows recordation of nucleotide incorporation in seconds.

In another embodiment, the present method comprises obtaining sequenceinformation for the nucleic acids in the test sample, e.g., cfDNA in amaternal test sample, using sequencing by hybridization.Sequencing-by-hybridization comprises contacting the plurality ofpolynucleotide sequences with a plurality of polynucleotide probes,wherein each of the plurality of polynucleotide probes can be optionallytethered to a substrate. The substrate might be flat surface comprisingan array of known nucleotide sequences. The pattern of hybridization tothe array can be used to determine the polynucleotide sequences presentin the sample. In other embodiments, each probe is tethered to a bead,e.g., a magnetic bead or the like. Hybridization to the beads can bedetermined and used to identify the plurality of polynucleotidesequences within the sample.

In another embodiment, the present method comprises obtaining sequenceinformation for the nucleic acids in the test sample, e.g., cfDNA in amaternal test sample, by massively parallel sequencing of millions ofDNA fragments using Illumina's sequencing-by-synthesis and reversibleterminator-based sequencing chemistry (e.g. as described in Bentley etal., Nature 6:53-59 [2009]). Template DNA can be genomic DNA, e.g.,cfDNA. In some embodiments, genomic DNA from isolated cells is used asthe template, and it is fragmented into lengths of several hundred basepairs. In other embodiments, cfDNA is used as the template, andfragmentation is not required as cfDNA exists as short fragments. Forexample fetal cfDNA circulates in the bloodstream as fragmentsapproximately 170 base pairs (bp) in length (Fan et al., Clin Chem56:1279-1286 [2010]), and no fragmentation of the DNA is required priorto sequencing. Illumina's sequencing technology relies on the attachmentof fragmented genomic DNA to a planar, optically transparent surface onwhich oligonucleotide anchors are bound. Template DNA is end-repaired togenerate 5′-phosphorylated blunt ends, and the polymerase activity ofKlenow fragment is used to add a single A base to the 3′ end of theblunt phosphorylated DNA fragments. This addition prepares the DNAfragments for ligation to oligonucleotide adapters, which have anoverhang of a single T base at their 3′ end to increase ligationefficiency. The adapter oligonucleotides are complementary to theflow-cell anchors. Under limiting-dilution conditions, adapter-modified,single-stranded template DNA is added to the flow cell and immobilizedby hybridization to the anchors. Attached DNA fragments are extended andbridge amplified to create an ultra-high density sequencing flow cellwith hundreds of millions of clusters, each containing ˜1,000 copies ofthe same template. In one embodiment, the randomly fragmented genomicDNA, e.g., cfDNA, is amplified using PCR before it is subjected tocluster amplification. Alternatively, an amplification-free genomiclibrary preparation is used, and the randomly fragmented genomic DNA,e.g., cfDNA is enriched using the cluster amplification alone (Kozarewaet al., Nature Methods 6:291-295 [2009]). The templates are sequencedusing a robust four-color DNA sequencing-by-synthesis technology thatemploys reversible terminators with removable fluorescent dyes.High-sensitivity fluorescence detection is achieved using laserexcitation and total internal reflection optics. Short sequence reads ofabout 20-40 bp, e.g., 36 bp, are aligned against a repeat-maskedreference genome and unique mapping of the short sequence reads to thereference genome are identified using specially developed data analysispipeline software. Non-repeat-masked reference genomes can also be used.Whether repeat-masked or non-repeat-masked reference genomes are used,only reads that map uniquely to the reference genome are counted. Aftercompletion of the first read, the templates can be regenerated in situto enable a second read from the opposite end of the fragments. Thus,either single-end or paired end sequencing of the DNA fragments can beused. Partial sequencing of DNA fragments present in the sample isperformed, and sequence tags comprising reads of predetermined length,e.g., 36 bp, are mapped to a known reference genome are counted. In oneembodiment, the reference genome sequence is the NCBI36/hg18 sequence,which is available on the world wide web atgenome.ucsc.edu/cgi-bin/hgGateway?org=Human&db=hg18&hgsid=166260105).Alternatively, the reference genome sequence is the GRCh37/hg19, whichis available on the world wide web at genome.ucsc.edu/cgi-bin/hgGateway.Other sources of public sequence information include GenBank, dbEST,dbSTS, EMBL (the European Molecular Biology Laboratory), and the DDBJ(the DNA Databank of Japan). A number of computer algorithms areavailable for aligning sequences, including without limitation BLAST(Altschul et al., 1990), BLITZ (MPsrch) (Sturrock & Collins, 1993),FASTA (Person & Lipman, 1988), BOWTIE (Langmead et al., Genome Biology10:R25.1-R25.10 [2009]), or ELAND (Illumina, Inc., San Diego, Calif.,USA). In one embodiment, one end of the clonally expanded copies of theplasma cfDNA molecules is sequenced and processed by bioinformaticalignment analysis for the Illumina Genome Analyzer, which uses theEfficient Large-Scale Alignment of Nucleotide Databases (ELAND)software.

In some embodiments of the methods described herein, the mapped sequencetags comprise sequence reads of about 20 bp, about 25 bp, about 30 bp,about 35 bp, about 40 bp, about 45 bp, about 50 bp, about 55 bp, about60 bp, about 65 bp, about 70 bp, about 75 bp, about 80 bp, about 85 bp,about 90 bp, about 95 bp, about 100 bp, about 110 bp, about 120 bp,about 130, about 140 bp, about 150 bp, about 200 bp, about 250 bp, about300 bp, about 350 bp, about 400 bp, about 450 bp, or about 500 bp. It isexpected that technological advances will enable single-end reads ofgreater than 500 bp enabling for reads of greater than about 1000 bpwhen paired end reads are generated. In one embodiment, the mappedsequence tags comprise sequence reads that are 36 bp. Mapping of thesequence tags is achieved by comparing the sequence of the tag with thesequence of the reference to determine the chromosomal origin of thesequenced nucleic acid (e.g. cfDNA) molecule, and specific geneticsequence information is not needed. A small degree of mismatch (0-2mismatches per sequence tag) may be allowed to account for minorpolymorphisms that may exist between the reference genome and thegenomes in the mixed sample.

A plurality of sequence tags are typically obtained per sample. In someembodiments, at least about 3×106 sequence tags, at least about 5×106sequence tags, at least about 8×106 sequence tags, at least about 10×106sequence tags, at least about 15×106 sequence tags, at least about20×106 sequence tags, at least about 30×106 sequence tags, at leastabout 40×106 sequence tags, or at least about 50×106 sequence tagscomprising between 20 and 40 bp reads, e.g., 36 bp, are obtained frommapping the reads to the reference genome per sample. In one embodiment,all the sequence reads are mapped to all regions of the referencegenome. In one embodiment, the tags that have been mapped to allregions, e.g., all chromosomes, of the reference genome are counted, andthe CNV, i.e., the over- or under-representation of a sequence ofinterest, e.g., a chromosome or portion thereof, in the mixed DNA sampleis determined. The method does not require differentiation between thetwo genomes.

The accuracy required for correctly determining whether a CNV, e.g.,aneuploidy, is present or absent in a sample, is predicated on thevariation of the number of sequence tags that map to the referencegenome among samples within a sequencing run (inter-chromosomalvariability), and the variation of the number of sequence tags that mapto the reference genome in different sequencing runs (inter-sequencingvariability). For example, the variations can be particularly pronouncedfor tags that map to GC-rich or GC-poor reference sequences. Othervariations can result from using different protocols for the extractionand purification of the nucleic acids, the preparation of the sequencinglibraries, and the use of different sequencing platforms. The presentmethod uses sequence doses (chromosome doses, or segment doses) based onthe knowledge of normalizing sequences (normalizing chromosome sequencesor normalizing segment sequences), to intrinsically account for theaccrued variability stemming from interchromosomal (intra-run), andinter-sequencing (inter-run) and platform-dependent variability.Chromosome doses are based on the knowledge of a normalizing chromosomesequence, which can be composed of a single chromosome, or of two ormore chromosomes selected from chromosomes 1-22, X, and Y.Alternatively, normalizing chromosome sequences can be composed of asingle chromosome segment, or of two or more segments of one chromosomeor of two or more chromosomes. Segment doses are based on the knowledgeof a normalizing segment sequence, which can be composed of a singlesegment of any one chromosome, or of two or more segments of any two ormore of chromosomes 1-22, X, and Y.

Apparatus and Systems for Determining Sequence of Interest

Analysis of the sequencing data and the diagnosis derived therefrom aretypically performed using various computer executed algorithms andprograms. Therefore, certain embodiments employ processes involving datastored in or transferred through one or more computer systems or otherprocessing systems. Embodiments disclosed herein also relate toapparatus for performing these operations. This apparatus may bespecially constructed for the required purposes, or it may be ageneral-purpose computer (or a group of computers) selectively activatedor reconfigured by a computer program and/or data structure stored inthe computer. In some embodiments, a group of processors performs someor all of the recited analytical operations collaboratively (e.g., via anetwork or cloud computing) and/or in parallel. A processor or group ofprocessors for performing the methods described herein may be of varioustypes including microcontrollers and microprocessors such asprogrammable devices (e.g., CPLDs and FPGAs) and non-programmabledevices such as gate array ASICs or general purpose microprocessors.

In addition, certain embodiments relate to tangible and/ornon-transitory computer readable media or computer program products thatinclude program instructions and/or data (including data structures) forperforming various computer-implemented operations. Examples ofcomputer-readable media include, but are not limited to, semiconductormemory devices, magnetic media such as disk drives, magnetic tape,optical media such as CDs, magneto-optical media, and hardware devicesthat are specially configured to store and perform program instructions,such as read-only memory devices (ROM) and random access memory (RAM).The computer readable media may be directly controlled by an end user orthe media may be indirectly controlled by the end user. Examples ofdirectly controlled media include the media located at a user facilityand/or media that are not shared with other entities. Examples ofindirectly controlled media include media that is indirectly accessibleto the user via an external network and/or via a service providingshared resources such as the “cloud.” Examples of program instructionsinclude both machine code, such as produced by a compiler, and filescontaining higher level code that may be executed by the computer usingan interpreter.

In various embodiments, the data or information employed in thedisclosed methods and apparatus is provided in an electronic format.Such data or information may include reads and tags derived from anucleic acid sample, counts or densities of such tags that align withparticular regions of a reference sequence (e.g., that align to achromosome or chromosome segment), reference sequences (includingreference sequences providing solely or primarily polymorphisms),chromosome and segment doses, calls such as aneuploidy calls, normalizedchromosome and segment values, pairs of chromosomes or segments andcorresponding normalizing chromosomes or segments, counselingrecommendations, diagnoses, and the like. As used herein, data or otherinformation provided in electronic format is available for storage on amachine and transmission between machines. Conventionally, data inelectronic format is provided digitally and may be stored as bits and/orbytes in various data structures, lists, databases, etc. The data may beembodied electronically, optically, etc.

One embodiment provides a computer program product for determining oneor more conditions of a fetus related to a sequence of interest. Thecomputer product may contain instructions for performing any one or moreof the above-described methods for determining a sequence of interestfor the fetus. As explained, the computer product may include anon-transitory and/or tangible computer readable medium having acomputer executable or compilable logic (e.g., instructions) recordedthereon for enabling a processor to determine chromosome doses and, insome cases, whether a fetal aneuploidy is present or absent. In oneexample, the computer product comprises (1) a computer readable mediumhaving a computer executable or compilable logic (e.g., instructions)recorded thereon for enabling a processor to diagnose a fetal aneuploidycomprising: a receiving procedure for receiving sequencing data from atleast a portion of nucleic acid molecules from a maternal biologicalsample, wherein said sequencing data comprises a calculated sequence tagcounts for libraries obtained from maternal cellular DNA and cfDNA; (2)computer assisted logic for analyzing a sequence of interest from saidreceived data; and (3) an output procedure for generating an outputindicating the conditions related to the sequence of interest.

It should be understood that it is not practical, or even possible inmost cases, for an unaided human being to perform the computationaloperations of the methods disclosed herein. For example, mapping asingle 30 bp read from a sample to any one of the human chromosomesmight require years of effort without the assistance of a computationalapparatus. Of course, the problem is compounded because reliableaneuploidy calls generally require mapping thousands (e.g., at leastabout 10,000) or even millions of reads to one or more chromosomes.

The methods disclosed herein can be performed using a system forevaluating a genetic sequence of interest for a fetus using cellular andcell free DNA from the mother. The system comprising: (a) a sequencerfor receiving nucleic acids from the test sample providing nucleic acidsequence information from the sample; (b) a processor; and (c) one ormore computer-readable storage media having stored thereon instructionsfor execution on said processor to evaluate sequence of interest for afetus using cellular and cell free DNA from the mother. The instructionscan specify one or more of the methods set forth herein. Accordingly, asystem of the present disclosure can provide an automated system forcarrying out a method set forth herein.

In some embodiments, the methods are instructed by a computer-readablemedium having stored thereon computer-readable instructions for carryingout a method for determining conditions of a fetus related to a sequenceof interest, e.g. carrying two copies of a disease causing allele. Thusone embodiment provides a computer program product comprising one ormore computer-readable non-transitory storage media having storedthereon computer-executable instructions that, when executed by one ormore processors of a computer system, cause the computer system toimplement a method for determining a condition of a fetus related to asequence of interest. The method includes: (a) obtaining sequence readsof mother-only cellular DNA obtained from the mother carrying the fetus,the cellular DNA having been specifically enriched for a sequence ofinterest; (b) computing a count of sequence tags mapping to the sequenceof interest for the cellular DNA; (c) obtaining sequence reads ofmother-and-fetus mixed cfDNA obtained from the mother, the cfDNA havingbeen specifically enriched for the sequence of interest; (d) computing acount of sequence tags mapping to the sequence of interest for thecfDNA; (e) comparing the sequence tag counts mapping to the sequence ofinterest between the cellular DNA and the cfDNA; and (f) determining thecondition of the fetus related to the sequence of interest.

In some embodiments, the method includes: obtaining sequence reads offetal cellular DNA obtained from the mother carrying the fetus;computing a count of sequence tags mapping to the sequence of interestfor the fetal cellular DNA; obtaining sequence reads of mother-and-fetusmixed cfDNA obtained from the mother; computing a count of sequence tagsmapping to the sequence of interest for the mixed cfDNA; and determiningthe condition of the fetus related to the sequence of interest based onthe sequence tag counts mapping to the sequence of interest for thefetal cellular DNA and the mixed cfDNA.

In some embodiments, the instructions may further include automaticallyrecording information pertinent to the method such as fetal fraction andthe presence or absence of a genetic disorder in a patient medicalrecord for a human subject providing the maternal test sample. Thepatient medical record may be maintained by, for example, a laboratory,physician's office, a hospital, a health maintenance organization, aninsurance company, or a personal medical record website. Further, basedon the results of the processor-implemented analysis, the method mayfurther involve prescribing, initiating, and/or altering treatment of ahuman subject from whom the maternal test sample was taken. This mayinvolve performing one or more additional tests or analyses onadditional samples taken from the subject.

Disclosed methods can also be performed using a computer processingsystem which is adapted or configured to perform a method fordetermining a fetal condition related to a sequence of interest, e.g., agenetic disorder or a fetal fraction. One embodiment provides a computerprocessing system which is adapted or configured to perform a method asdescribed herein. In one embodiment, the apparatus comprises asequencing device adapted or configured for sequencing at least aportion of the nucleic acid molecules in a sample to obtain the type ofsequence information described elsewhere herein. The apparatus may alsoinclude components for processing the sample. Such components aredescribed elsewhere herein.

Sequence or other data, can be input into a computer or stored on acomputer readable medium either directly or indirectly. In oneembodiment, a computer system is directly coupled to a sequencing devicethat reads and/or analyzes sequences of nucleic acids from samples.Sequences or other information from such tools are provided viainterface in the computer system. Alternatively, the sequences processedby system are provided from a sequence storage source such as a databaseor other repository. Once available to the processing apparatus, amemory device or mass storage device buffers or stores, at leasttemporarily, sequences of the nucleic acids. In addition, the memorydevice may store tag counts for various chromosomes or genomes, etc. Thememory may also store various routines and/or programs for analyzing thepresenting the sequence or mapped data. Such programs/routines mayinclude programs for performing statistical analyses, etc.

In one example, a user provides a sample into a sequencing apparatus.Data is collected and/or analyzed by the sequencing apparatus which isconnected to a computer. Software on the computer allows for datacollection and/or analysis. Data can be stored, displayed (via a monitoror other similar device), and/or sent to another location. The computermay be connected to the internet which is used to transmit data to ahandheld device utilized by a remote user (e.g., a physician, scientistor analyst). It is understood that the data can be stored and/oranalyzed prior to transmittal. In some embodiments, raw data iscollected and sent to a remote user or apparatus that will analyzeand/or store the data. Transmittal can occur via the internet, but canalso occur via satellite or other connection. Alternately, data can bestored on a computer-readable medium and the medium can be shipped to anend user (e.g., via mail). The remote user can be in the same or adifferent geographical location including, but not limited to abuilding, city, state, country or continent.

In some embodiments, the methods also include collecting data regardinga plurality of polynucleotide sequences (e.g., reads, tags and/orreference chromosome sequences) and sending the data to a computer orother computational system. For example, the computer can be connectedto laboratory equipment, e.g., a sample collection apparatus, anucleotide amplification apparatus, a nucleotide sequencing apparatus,or a hybridization apparatus. The computer can then collect applicabledata gathered by the laboratory device. The data can be stored on acomputer at any step, e.g., while collected in real time, prior to thesending, during or in conjunction with the sending, or following thesending. The data can be stored on a computer-readable medium that canbe extracted from the computer. The data collected or stored can betransmitted from the computer to a remote location, e.g., via a localnetwork or a wide area network such as the internet. At the remotelocation various operations can be performed on the transmitted data asdescribed below.

Among the types of electronically formatted data that may be stored,transmitted, analyzed, and/or manipulated in systems, apparatus, andmethods disclosed herein are the following:

-   -   Reads obtained by sequencing nucleic acids in a test sample;    -   Tracking information that correlates indexes with the identity        of samples or subfractions of samples;    -   Tags obtained by aligning reads to a reference genome or other        reference sequence or sequences;    -   The reference genome or sequence;    -   Sequence tag density—Counts or numbers of tags for each of two        or more regions (typically chromosomes or chromosome segments)        of a reference genome or other reference sequences;    -   Diagnoses (clinical condition associated with the calls);    -   Recommendations for further tests derived from the calls and/or        diagnoses;    -   Quality metrics for samples, sequence data derived therefrom or        diagnoses; and/or    -   Treatment and/or monitoring plans derived from the calls and/or        diagnoses.

These various types of data may be obtained, stored transmitted,analyzed, and/or manipulated at one or more locations using distinctapparatus. The processing options span a wide spectrum. At one end ofthe spectrum, all or much of this information is stored and used at thelocation where the test sample is processed, e.g., a doctor's office orother clinical setting. In other extreme, the sample is obtained at onelocation, it is processed and optionally sequenced at a differentlocation, reads are aligned and calls are made at one or more differentlocations, and diagnoses, recommendations, and/or plans are prepared atstill another location (which may be a location where the sample wasobtained).

In various embodiments, the reads are generated with the sequencingapparatus and then transmitted to a remote site where they are processedto determine conditions related to the sequence of interest. At thisremote location, as an example, the reads are aligned to a referencesequence to produce tags, which are counted and assigned to chromosomesor segments of interest. Also at the remote location, the counts areconverted to doses using associated normalizing chromosomes or segments.Still further, at the remote location, the doses are used to generateaneuploidy calls.

Among the processing operations that may be employed at distinctlocations are the following:

-   -   Sample collection;    -   Sample processing preliminary to sequencing;    -   Sequencing;    -   Analyzing sequence data and deriving aneuploidy calls;    -   Diagnosis;    -   Reporting a diagnosis and/or a call to patient or health care        provider;    -   Developing a plan for further treatment, testing, and/or        monitoring;    -   Executing the plan; and/or    -   Counseling.

Any one or more of these operations may be automated as describedelsewhere herein. Typically, the sequencing and the analyzing ofsequence data and deriving aneuploidy calls will be performedcomputationally. The other operations may be performed manually orautomatically.

Examples of locations where sample collection may be performed includehealth practitioners' offices, clinics, patients' homes (where a samplecollection tool or kit is provided), and mobile health care vehicles.Examples of locations where sample processing prior to sequencing may beperformed include health practitioners' offices, clinics, patients'homes (where a sample processing apparatus or kit is provided), mobilehealth care vehicles, and facilities of aneuploidy analysis providers.Examples of locations where sequencing may be performed include healthpractitioners' offices, clinics, health practitioners' offices, clinics,patients' homes (where a sample sequencing apparatus and/or kit isprovided), mobile health care vehicles, and facilities of aneuploidyanalysis providers. The location where the sequencing takes place may beprovided with a dedicated network connection for transmitting sequencedata (typically reads) in an electronic format. Such connection may bewired or wireless and have and may be configured to send the data to asite where the data can be processed and/or aggregated prior totransmission to a processing site. Data aggregators can be maintained byhealth organizations such as Health Maintenance Organizations (HMOs).

The analyzing and/or deriving operations may be performed at any of theforegoing locations or alternatively at a further remote site dedicatedto computation and/or the service of analyzing nucleic acid sequencedata. Such locations include for example, clusters such as generalpurpose server farms, the facilities of an aneuploidy analysis servicebusiness, and the like. In some embodiments, the computational apparatusemployed to perform the analysis is leased or rented. The computationalresources may be part of an internet accessible collection of processorssuch as processing resources colloquially known as the cloud. In somecases, the computations are performed by a parallel or massivelyparallel group of processors that are affiliated or unaffiliated withone another. The processing may be accomplished using distributedprocessing such as cluster computing, grid computing, and the like. Insuch embodiments, a cluster or grid of computational resourcescollective form a super virtual computer composed of multiple processorsor computers acting together to perform the analysis and/or derivationdescribed herein. These technologies as well as more conventionalsupercomputers may be employed to process sequence data as describedherein. Each is a form of parallel computing that relies on processorsor computers. In the case of grid computing these processors (oftenwhole computers) are connected by a network (private, public, or theInternet) by a conventional network protocol such as Ethernet. Bycontrast, a supercomputer has many processors connected by a localhigh-speed computer bus.

In certain embodiments, the diagnosis (e.g., the fetus has Downssyndrome or the patient has a particular type of cancer) is generated atthe same location as the analyzing operation. In other embodiments, itis performed at a different location. In some examples, reporting thediagnosis is performed at the location where the sample was taken,although this need not be the case. Examples of locations where thediagnosis can be generated or reported and/or where developing a plan isperformed include health practitioners' offices, clinics, internet sitesaccessible by computers, and handheld devices such as cell phones,tablets, smart phones, etc. having a wired or wireless connection to anetwork. Examples of locations where counseling is performed includehealth practitioners' offices, clinics, internet sites accessible bycomputers, handheld devices, etc.

In some embodiments, the sample collection, sample processing, andsequencing operations are performed at a first location and theanalyzing and deriving operation is performed at a second location.However, in some cases, the sample collection is collected at onelocation (e.g., a health practitioner's office or clinic) and the sampleprocessing and sequencing is performed at a different location that isoptionally the same location where the analyzing and deriving takeplace.

In various embodiments, a sequence of the above-listed operations may betriggered by a user or entity initiating sample collection, sampleprocessing and/or sequencing. After one or more these operations havebegun execution the other operations may naturally follow. For example,the sequencing operation may cause reads to be automatically collectedand sent to a processing apparatus which then conducts, oftenautomatically and possibly without further user intervention, thesequence analysis and derivation of aneuploidy operation. In someimplementations, the result of this processing operation is thenautomatically delivered, possibly with reformatting as a diagnosis, to asystem component or entity that processes reports the information to ahealth professional and/or patient. As explained such information canalso be automatically processed to produce a treatment, testing, and/ormonitoring plan, possibly along with counseling information. Thus,initiating an early stage operation can trigger an end to end sequencein which the health professional, patient or other concerned party isprovided with a diagnosis, a plan, counseling and/or other informationuseful for acting on a physical condition. This is accomplished eventhough parts of the overall system are physically separated and possiblyremote from the location of, e.g., the sample and sequence apparatus.

FIG. 15 shows one implementation of a dispersed system for producing acall or diagnosis from a test sample. A sample collection location 01 isused for obtaining a test sample from a patient such as a pregnantfemale or a putative cancer patient. The samples then provided to aprocessing and sequencing location 03 where the test sample may beprocessed and sequenced as described above. Location 03 includesapparatus for processing the sample as well as apparatus for sequencingthe processed sample. The result of the sequencing, as describedelsewhere herein, is a collection of reads which are typically providedin an electronic format and provided to a network such as the Internet,which is indicated by reference number 05 in FIG. 15.

The sequence data is provided to a remote location 07 where analysis andcall generation are performed. This location may include one or morepowerful computational devices such as computers or processors. Afterthe computational resources at location 07 have completed their analysisand generated a call from the sequence information received, the call isrelayed back to the network 05. In some implementations, not only is acall generated at location 07 but an associated diagnosis is alsogenerated. The call and or diagnosis are then transmitted across thenetwork and back to the sample collection location 01 as illustrated inFIG. 15. As explained, this is simply one of many variations on how thevarious operations associated with generating a call or diagnosis may bedivided among various locations. One common variant involves providingsample collection and processing and sequencing in a single location.Another variation involves providing processing and sequencing at thesame location as analysis and call generation.

FIG. 16 elaborates on the options for performing various operations atdistinct locations. In the most granular sense depicted in FIG. 16, eachof the following operations is performed at a separate location: samplecollection, sample processing, sequencing, read alignment, calling,diagnosis, and reporting and/or plan development.

In one embodiment that aggregates some of these operations, sampleprocessing and sequencing are performed in one location and readalignment, calling, and diagnosis are performed at a separate location.See the portion of FIG. 16 identified by reference character A. Inanother implementation, which is identified by character B in FIG. 16,sample collection, sample processing, and sequencing are all performedat the same location. In this implementation, read alignment and callingare performed in a second location. Finally, diagnosis and reportingand/or plan development are performed in a third location. In theimplementation depicted by character C in FIG. 16, sample collection isperformed at a first location, sample processing, sequencing, readalignment, calling, and diagnosis are all performed together at a secondlocation, and reporting and/or plan development are performed at a thirdlocation. Finally, in the implementation labeled D in FIG. 16, samplecollection is performed at a first location, sample processing,sequencing, read alignment, and calling are all performed at a secondlocation, and diagnosis and reporting and/or plan management areperformed at a third location.

One embodiment provides a system for use in determining a condition of afetus related to a sequence of interest using maternal-only nucleic acid(e.g. maternal cellular DNA) and mixed maternal-fetal nucleic acid (e.g.cfDNA) from a maternal sample, the system including a sequencer forreceiving a nucleic acid sample and providing fetal and maternal nucleicacid sequence information from the sample; a processor; and a machinereadable storage medium comprising instructions for execution on saidprocessor, the instructions comprising: (a) code for obtaining sequencereads of maternal-only nucleic acid obtained from the mother carryingthe fetus, the maternal-only nucleic acid having been optionallyenriched for a specific sequence of interest; (b) code for computing acount of sequence tags mapping to the sequence of interest for thematernal-only nucleic acid; (c) code for obtaining sequence reads ofmaternal-fetal mixed nucleic acid obtained from the mother, thematernal-fetal mixed nucleic acid having, optionally, been specificallyenriched for the sequence of interest; (d) code for computing a count ofsequence tags mapping to the sequence of interest for the maternal-fetalmixed nucleic acid; (e) code for comparing the sequence tag countsmapping to the sequence of interest between the cellular maternal-onlynucleic acid and the maternal-fetal mixed nucleic acid; and (f) code fordetermining the condition of the fetus related to the sequence ofinterest.

In some alternative embodiments, the instructions comprise: (a) code forobtaining sequence reads of fetal cellular DNA obtained from the mothercarrying the fetus; (b) code for computing a count of sequence tagsmapping to the sequence of interest for the fetal cellular DNA; (c) codefor obtaining sequence reads of mother-and-fetus mixed cfDNA obtainedfrom the mother; (d) code for computing a count of sequence tags mappingto the sequence of interest for the mixed cfDNA; and (e) code fordetermining the condition of the fetus related to the sequence ofinterest based on the sequence tag counts mapping to the sequence ofinterest for the fetal cellular DNA and the mixed cfDNA.

In some embodiments of any of the systems provided herein, the sequenceris configured to perform next generation sequencing (NGS). In someembodiments, the sequencer is configured to perform massively parallelsequencing using sequencing-by-synthesis with reversible dyeterminators. In other embodiments, the sequencer is configured toperform sequencing-by-ligation. In yet other embodiments, the sequenceris configured to perform single molecule sequencing.

Example 1

Preparation and Sequencing of Primary and Enriched Sequencing Libraries

a. Preparation of Sequencing Libraries—Abbreviated Protocol (ABB)

All sequencing libraries, i.e., primary and enriched libraries, wereprepared from approximately 2 ng of purified cfDNA that was extractedfrom maternal plasma. Library preparation was performed using reagentsof the NEBNext™ DNA Sample Prep DNA Reagent Set 1 (Part No. E6000L; NewEngland Biolabs, Ipswich, Mass.), for Illumina® as follows. Becausecell-free plasma DNA is fragmented in nature, no further fragmentationby nebulization or sonication was done on the plasma DNA samples. Theoverhangs of approximately 2 ng purified cfDNA fragments contained in 40μl were converted into phosphorylated blunt ends according to theNEBNext® End Repair Module by incubating in a 1.5 ml microfuge tube thecfDNA with 5 μl 10× phosphorylation buffer, 2 μl deoxynucleotidesolution mix (10 mM each dNTP), 1 μl of a 1:5 dilution of DNA PolymeraseI, 1 μl T4 DNA Polymerase and 1 μl T4 Polynucleotide Kinase provided inthe NEBNext™ DNA Sample Prep DNA Reagent Set 1 for 15 minutes at 20° C.The enzymes were then heat inactivated by incubating the reactionmixture at 75° C. for 5 minutes. The mixture was cooled to 4° C., and dAtailing of the blunt-ended DNA was accomplished using 10 μl of thedA-tailing master mix containing the Klenow fragment (3′ to 5′ exominus) (NEBNext™ DNA Sample Prep DNA Reagent Set 1), and incubating for15 minutes at 37° C. Subsequently, the Klenow fragment was heatinactivated by incubating the reaction mixture at 75° C. for 5 minutes.Following the inactivation of the Klenow fragment, 1 μl of a 1:5dilution of Illumina Genomic Adaptor Oligo Mix (Part No. 1000521;Illumina Inc., Hayward, Calif.) was used to ligate the Illumina adaptors(Non-Index Y-Adaptors) to the dA-tailed DNA using 4 μl of the T4 DNAligase provided in the NEBNext™ DNA Sample Prep DNA Reagent Set 1, byincubating the reaction mixture for 15 minutes at 25° C. The mixture wascooled to 4° C., and the adaptor-ligated cfDNA was purified fromunligated adaptors, adaptor dimers, and other reagents using magneticbeads provided in the Agencourt AMPure XP PCR purification system (PartNo. A63881; Beckman Coulter Genomics, Danvers, Mass.). Eighteen cyclesof PCR were performed to selectively enrich adaptor-ligated cfDNA (25μl) using Phusion® High-Fidelity Master Mix (25 μl; Finnzymes, Woburn,Mass.) and Illumina's PCR primers (0.5 μM each) complementary to theadaptors (Part No. 1000537 and 1000537). The adaptor-ligated DNA wassubjected to PCR (98° C. for 30 seconds; 18 cycles of 98° C. for 10seconds, 65° C. for 30 seconds, and 72° C. for 30; final extension at72° C. for 5 minutes, and hold at 4° C.) using Illumina Genomic PCRPrimers (Part Nos. 100537 and 1000538) and the Phusion HF PCR Master Mixprovided in the NEBNext™ DNA Sample Prep DNA Reagent Set 1, according tothe manufacturer's instructions. The amplified product was purifiedusing the Agencourt AMPure XP PCR purification system (AgencourtBioscience Corporation, Beverly, Mass.) according to the manufacturer'sinstructions available atwww.beckmangenomics.com/products/AMPureXPProtocol_000387v001.pdf. Thepurified amplified product was eluted in 40 μl of Qiagen EB Buffer, andthe concentration and size distribution of the amplified libraries wasanalyzed using the Agilent DNA 1000 Kit for the 2100 Bioanalyzer(Agilent technologies Inc., Santa Clara, Calif.).

b. Preparation of Sequencing Libraries Full-Length Protocol

The full-length protocol described here is essentially the standardprotocol provided by Illumina, and only differs from the Illuminaprotocol in the purification of the amplified library. The Illuminaprotocol instructs that the amplified library be purified using gelelectrophoresis, while the protocol described herein uses magnetic beadsfor the same purification step. Approximately 2 ng of purified cfDNAextracted from maternal plasma was used to prepare a primary sequencinglibrary using NEBNext™ DNA Sample Prep DNA Reagent Set 1 (Part No.E6000L; New England Biolabs, Ipswich, Mass.) for Illumina® essentiallyaccording to the manufacturer's instructions. All steps except for thefinal purification of the adaptor-ligated products, which was performedusing Agencourt magnetic beads and reagents instead of the purificationcolumn, were performed according to the protocol accompanying theNEBNext™ Reagents for Sample Preparation for a genomic DNA library thatis sequenced using the Illumina® GAIL The NEBNext™ protocol essentiallyfollows that provided by Illumina, which is available atgrcf.jhml.edu/hts/protocols/11257047_ChIP_Sample_Prep.pdf.

The overhangs of approximately 2 ng purified cfDNA fragments containedin 40 μl were converted into phosphorylated blunt ends according to theNEBNext® End Repair Module by incubating the 40 μl cfDNA with 5 μl 10×phosphorylation buffer, 2 μl deoxynucleotide solution mix (10 mM eachdNTP), 1 μl A of a 1:5 dilution of DNA Polymerase I, 1 μl T4 DNAPolymerase and 1 μl T4 Polynucleotide Kinase provided in the NEBNext™DNA Sample Prep DNA Reagent Set 1 in a 200 μl microfuge tube in athermal cycler for 30 minutes at 20° C. The sample was cooled to 4° C.,and purified using a QIAQuick column provided in the QIAQuick PCRPurification Kit (QIAGEN Inc., Valencia, Calif.) as follows. The 50 μlreaction was transferred to 1.5 ml microfuge tube, and 250 μl of QiagenBuffer PB were added. The resulting 300 μl were transferred to aQIAquick column, which was centrifuged at 13,000 RPM for 1 minute in amicrofuge. The column was washed with 750 μl Qiagen Buffer PE, andre-centrifuged. Residual ethanol was removed by an additionalcentrifugation for 5 minutes at 13,000 RPM. The DNA was eluted in 39 μlQiagen Buffer EB by centrifugation. dA tailing of 34 μl of theblunt-ended DNA was accomplished using 16 μl of the dA-tailing mastermix containing the Klenow fragment (3′ to 5′ exo minus) (NEBNext™ DNASample Prep DNA Reagent Set 1), and incubating for 30 minutes at 37° C.according to the manufacturer's NEBNext® dA-Tailing Module. The samplewas cooled to 4° C., and purified using a column provided in theMinElute PCR Purification Kit (QIAGEN Inc., Valencia, Calif.) asfollows. The 50 μl reaction was transferred to 1.5 ml microfuge tube,and 250 μl of Qiagen Buffer PB were added. The 300 μl were transferredto the MinElute column, which was centrifuged at 13,000 RPM for 1 minutein a microfuge. The column was washed with 750 μl Qiagen Buffer PE, andre-centrifuged. Residual ethanol was removed by an additionalcentrifugation for 5 minutes at 13,000 RPM. The DNA was eluted in 15 μlQiagen Buffer EB by centrifugation. Ten microliters of the DNA eluatewere incubated with 1 μl of a 1:5 dilution of the Illumina GenomicAdapter Oligo Mix (Part No. 1000521), 15 μl of 2× Quick LigationReaction Buffer, and 4 μl Quick T4 DNA Ligase, for 15 minutes at 25° C.according to the NEBNext® Quick Ligation Module. The sample was cooledto 4° C., and purified using a MinElute column as follows. One hundredand fifty microliters of Qiagen Buffer PE were added to the 30 μlreaction, and the entire volume was transferred to a MinElute columnwere transferred to a MinElute column, which was centrifuged at 13,000RPM for 1 minute in a microfuge. The column was washed with 750 μlQiagen Buffer PE, and re-centrifuged. Residual ethanol was removed by anadditional centrifugation for 5 minutes at 13,000 RPM. The DNA waseluted in 28 μl Qiagen Buffer EB by centrifugation. Twenty threemicroliters of the adaptor-ligated DNA eluate were subjected to 18cycles of PCR (98° C. for 30 seconds; 18 cycles of 98° C. for 10seconds, 65° C. for 30 seconds, and 72° C. for 30; final extension at72° C. for 5 minutes, and hold at 4° C.) using Illumina Genomic PCRPrimers (Part Nos. 100537 and 1000538) and the Phusion HF PCR Master Mixprovided in the NEBNext™ DNA Sample Prep DNA Reagent Set 1, according tothe manufacturer's instructions. The amplified product was purifiedusing the Agencourt AMPure XP PCR purification system (AgencourtBioscience Corporation, Beverly, Mass.) according to the manufacturer'sinstructions available atwww.beckmangenomics.com/products/AMPureXPProtocol_000387v001.pdf. TheAgencourt AMPure XP PCR purification system removes unincorporateddNTPs, primers, primer dimers, salts and other contaminates, andrecovers amplicons greater than 100 bp. The purified amplified productwas eluted from the Agencourt beads in 40 μl of Qiagen EB Buffer and thesize distribution of the libraries was analyzed using the Agilent DNA1000 Kit for the 2100 Bioanalyzer (Agilent technologies Inc., SantaClara, Calif.).

c. Analysis of Sequencing Libraries Prepared According to theAbbreviated (a) and the Full-Length (b) Protocols

The electropherograms generated by the Bioanalyzer are shown in FIGS.17A and 17B. FIG. 17A shows the electropherogram of library DNA preparedfrom cfDNA purified from plasma sample M24228 using the full-lengthprotocol described in (a), and FIG. 17B shows the electropherogram oflibrary DNA prepared from cfDNA purified from plasma sample M24228 usingthe full-length protocol described in (b). In both figures, peaks 1 and4 represent the 15 bp Lower Marker, and the 1,500 Upper Marker,respectively; the numbers above the peaks indicate the migration timesfor the library fragments; and the horizontal lines indicate the setthreshold for integration. The electropherogram in FIG. 17A shows aminor peak of fragments of 187 bp and a major peak of fragments of 263bp, while the electropherogram in FIG. 17B shows only one peak at 265bp. Integration of the peak areas resulted in a calculated concentrationof 0.40 ng/μl for the DNA of the 187 bp peak in FIG. 17A, aconcentration of 7.34 ng/μl for the DNA of the 263 bp peak in FIG. 17A,and a concentration of 14.72 ng/μl for the DNA of the 265 bp peak inFIG. 17B. The Illumina adaptors that were ligated to the cfDNA are knownto be 92 bp, which when subtracted from the 265 bp, indicate that thepeak size of the cfDNA is 173 bp. It is possible that the minor peak at187 bp represents fragments of two primers that were ligated end-to-end.The linear two-primer fragments are eliminated from the final libraryproduct when the abbreviated protocol is used. The abbreviated protocolalso eliminates other smaller fragments of less than 187 bp. In thisexample, the concentration of purified adaptor-ligated cfDNA is doublethat of the adaptor-ligated cfDNA produced using the full-lengthprotocol. It has been noted that the concentration of theadaptor-ligated cfDNA fragments was always greater than that obtainedusing the full-length protocol (data not shown).

Thus, an advantage of preparing the sequencing library using theabbreviated protocol is that the library obtained consistently comprisesonly one major peak in the 262-267 bp range while the quality of thelibrary prepared using the full-length protocol varies as reflected bythe number and mobility of peaks other than that representing the cfDNA.Non-cfDNA products would occupy space on the flow cell and diminish thequality of the cluster amplification and subsequent imaging of thesequencing reactions, which underlies the overall assignment of theaneuploidy status. The abbreviated protocol was shown not to affect thesequencing of the library.

Another advantage of preparing the sequencing library using theabbreviated protocol is that the three enzymatic steps of blunt-ending,d-A tailing, and adaptor-ligation, take less than an hour to complete tosupport the validation and implementation of a rapid aneuploiddiagnostic service.

Another advantage is that the three enzymatic steps of blunt-ending, d-Atailing, and adaptor ligation, are performed in the same reaction tube,thus avoiding multiple sample transfers that would potentially lead toloss of material, and more importantly to possible sample mix-up andsample contamination.

Example 2: Determining Sry Gene Using Fetal Cellular DNA andMother-and-Fetus cfDNA

This example illustrates that the methods and systems disclosed hereinmay be used to determine a Y-chromosome specific sry gene using cfDNAand fetal cellular DNA obtained from a maternal blood sample.

In this example, cfDNA are isolated and indexed cfDNA library areprepared from the isolated cfDNA. In this example, the plasma or serumfrom maternal blood sample was harvested by a low-speed centrifugationat 1,600 g. A maternal blood sample was spin for 15 mins at 4° C. Thenthe upper layer of plasma was removed, then the plasma was spun 2 timesfor 10 mins. at 16,000 g to ensure removal of all cellular sources ofDNA. Then Tn5 mediated transposon tagging (Nextera) was used to createan indexed sequencing library directly from 15 ul of prespun plasma orserum (or kit purified cell free DNA). NGS libraries prepared frompurified cfDNA as shown in are shown in FIG. 18.

In addition, circulating fetal NRBCs are isolated using a magneticsweeping device which is capable of isolating circulating fetal cellsfrom maternal blood. This process for isolating live nucleated fetal redblood cells is outlined in the following steps: (a) labeling of allcellular nuclei in a maternal blood sample with Hoechst; (b)differential red blood lysis of maternal red blood cells—usingacetazolamide treatment to protect fetal nucleated red blood cells fromred blood cell lysis; (c) fetal nucleated red blood cells were labeledusing magnetic beads coated with an antibody that recognizes a cellsurface marker present on fetal red blood surfaces, and fluorescentlytagged using labeled antibodies to markers on the surfaces of fetal redblood cells. Preparations of fetal cells were then enriched using amagnetic sweeping device, and (d) imaging and isolation of fetal NRBCswas accomplished using an image guided cell isolation device.

Then, purified fetal cell were lysed and indexed library was preparedfrom the lysed fetal NRBCs. Single purified fetal NRBC or small pools ofcells (<100 cells) were lysed and their DNA was released by incubatingfor 6 min. in Quick Extract Buffer (Epicentre) followed by incubation at95° C. for 2 min to inactivate proteinase K in extraction buffer. Thenthe example used Tn5 mediated transposon tagging (Nextera) to create anindexed sequencing library directly from the fetal cell lysates. Theindex for the cellular DNA library is different from the index for themixed cfDNA.

The example involved mixing indexed cfDNA library and indexed purifiedfetal cell library, quantification, and clustering on a sequencingplatform by Illumina. Uniquely indexed cfDNA and fetal cell librarieswere mixed and the sample quantified using qPCR. Samples was clusteredon an Illumina flow cell using the cBot.

Then sequencing and bioinformatics were performed on the libraries. Flowcells were subjected to Illumina paired end sequencing. Qualityfiltering of NGS reads and index decoding was performed. Fetal read datawas aligned to reference chromosomes. Sequence reads were counted todetect chromosomal anueploidy in cfDNA and fetal cell DNA. Paired endread analysis was used to detect chromosomal rearrangements in fetalcell DNA. High resolution sequence analysis was performed, using indexedpurified fetal cell sequence to detect indels, copy number variation,SNPs and other sequence changes that were of diagnostic value.

Using qPCR for the Y chromosome specific gene sry, a strong correlationwas found between the presence of sry in cfDNA and isolated fetal cellsfrom the same sample when the fetus is male. FIG. 19 shows the dataobtained for 9 subjects. These data indicated that one can obtaininformation of diagnostic value from both cfDNA and isolated fetal cellsfrom the same sample.

The present disclosure may be embodied in other specific forms withoutdeparting from its spirit or essential characteristics. The describedembodiments are to be considered in all respects only as illustrativeand not restrictive. The scope of the disclosure is, therefore,indicated by the appended claims rather than by the foregoingdescription. All changes which come within the meaning and range ofequivalency of the claims are to be embraced within their scope.

What is claimed is:
 1. A method for determining at least one sequence ofinterest of a fetus of a pregnant mother, the method comprising: (a)obtaining fetal cellular DNA from the blood of the pregnant mother; (b)obtaining mother-and-fetus mixed cfDNA from the blood of the pregnantmother; (c) applying a first index sequence to the fetal cellular DNAand a second index sequence to the mixed cfDNA, wherein the first indexsequence and the second index sequence are different, the first indexsequence specifies a source of DNA as being from the fetal cellular DNAfrom the pregnant mother, and the second index sequence specifies asource of DNA as being from the mixed cfDNA from the pregnant mother;(d) combining the fetal cellular DNA and the mixed cfDNA to provide asample of combined cellular DNA and cfDNA; (e) sequencing the sample ofcombined cellular DNA and cfDNA to provide a plurality of sequence readsand mapping the plurality of sequence reads to a reference sequence; and(f) analyzing the mapped sequence reads to determine a presence and/orabundance of the at least one sequence of interest in the fetus's DNA,wherein at least a portion of the mapped sequence reads maps to the atleast one sequence of interest.
 2. The method of claim 1, wherein thefetal cellular DNA is obtained from one or more fetal nucleated redblood cells (NRBCs) in the blood of the pregnant mother.
 3. The methodof claim 2, wherein (a) comprises separating the fetal NRBCs frommaternal erythrocytes in a cellular component of a blood sample of thepregnant mother.
 4. The method of claim 3, wherein separating the fetalNRBCs from the maternal erythrocytes comprises differentially lysingmaternal erythrocytes.
 5. The method of claim 3, wherein separating thefetal NRBCs from the maternal erythrocytes comprises size-basedseparation and/or capture-based separation.
 6. The method of claim 5,wherein the capture-based separation comprises capturing the fetal NRBCsthrough binding one or more cellular markers expressed by fetal NRBCs.7. The method of claim 6, wherein the one or more cellular markersexpressed by fetal NRBCs are selected from the group consisting of CD71,CD36, CD34, antigen-i, galactose, glycophorin-A, fetal haemoglobin, andany combinations thereof.
 8. The method of claim 6, wherein the one ormore cellular markers comprise a surface marker expressed by fetal NRBCsbut not, or to a lesser degree, by maternal NRBCs.
 9. The method ofclaim 6, wherein the one or more cellular markers comprises a4B9-antigen and/or a 4B8-antigen.
 10. The method of claim 6, wherein thecapture-based separation comprise binding magnetically responsiveparticles to fetal NRBCs, wherein the magnetically responsive particleshave an affinity to one or more cellular markers expressed by fetalNRBCs.
 11. The method of claim 10, wherein the capture-based separationis performed by an automated immunomagnetic separation device.
 12. Themethod of claim 6, wherein the capture-based separation comprise bindingfluorescent labels to fetal NRBCs, wherein the fluorescent labels havean affinity to one or more cellular markers expressed by fetal NRBCs.13. The method of claim 1, further comprising: obtaining a blood samplefrom the pregnant mother; separating an erythrocyte fraction and aplasma fraction of the blood sample; obtaining the fetal cellular DNAfrom the erythrocyte fraction of the blood sample; and obtaining thecfDNA from the plasma fraction of the blood sample.
 14. The method ofclaim 1, further comprising preparing a first sequencing library of thefetal cellular DNA and a second sequencing library of the cfDNA.
 15. Themethod of claim 14, wherein applying the first and the second indexsequences comprises hybridizing and extending adapter oligonucleotidescomprising the index sequences.
 16. The method of claim 15, wherein theadapter oligonucleotides comprise locus-specific extensionoligonucleotides.
 17. The method of claim 16, wherein the locus-specificextension oligonucleotides are selective for two or more alleles of adisease related gene.
 18. The method of claim 15, wherein each of theadapter oligonucleotides comprises an adapter sequence or a portionthereof, wherein the adapter sequence is configured to hybridize to anoligonucleotide attached to a substrate of a flow cell of a sequencingapparatus.
 19. The method of claim 14, wherein incorporating indexes ineach of said sequencing libraries comprises ligating or transposingsequences comprising the index sequences to the fetal cellular DNA andthe mixed cfDNA.
 20. The method of claim 14, further comprisingincorporating an individual-specific index to the sequencing libraries,wherein the individual-specific index indicates an identity of thepregnant mother, thereby allowing the pregnant mother's DNA to beprocessed with other individuals' DNA for parallel sequencing.
 21. Themethod of claim 14, wherein the sequencing libraries are transposoninsertion libraries.
 22. The method of claim 1, further comprisingdetermining whether the fetus has a genetic disease from the at leastone sequence of interest of the fetus.
 23. The method of claim 1,wherein the at least one sequence of interest comprises a diseaseassociated allele selected from the group consisting: a singlenucleotide polymorphism, a tandem repeat, a micro-deletion, aninsertion, an indel, and any combinations thereof.
 24. The method ofclaim 1, further comprising determining a complete or partialaneuploidy.